TWI814219B - Glucagon-receptor selective polypeptides and methods of use thereof - Google Patents

Abstract

This invention relates to isolated polypeptides that are glucagon-receptor selective analogs and peptide derivatives thereof. These analogs are selective for human glucagon receptor with improved solubility, thermal stability, and physicochemical properties as compared to native endogenous glucagon. This invention also relates to methods of using such polypeptides in a variety of therapeutic and diagnostic indications, as well as methods of producing such polypeptides. These analogs are useful, alone or in combination with other therapeutic peptides, in methods of treating obesity, diabetes, metabolic disorders, and other disorders or disease.

Description

Glucagon receptor selective polypeptides and methods of using them [Related Applications]

This application claims U.S. Provisional Patent Application No. 62/337,005, filed on May 16, 2016; U.S. Provisional Patent Application No. 62/414,146, filed on October 28, 2016; and U.S. Provisional Patent Application No. 62/414,146, filed on November 11, 2016. priority to U.S. Provisional Patent Application No. 62/420,937, the entire contents of each of which are incorporated herein by reference.

The present invention relates to isolated polypeptides which are glucagon receptor selective analogs and peptide derivatives thereof. These analogs and peptide derivatives have improved solubility, thermal stability and physicochemical properties compared to natural endogenous glucagon. The invention also relates to methods of using such polypeptides for various therapeutic and diagnostic indications and methods of producing such polypeptides. These analogs are useful in methods of treating obesity, diabetes, metabolic disorders, and other diseases or conditions.

Glucagon, a peptide hormone produced by pancreatic alpha cells, and glucagon-like peptide-1 (GLP-1, a neuropeptide) are derived from pre-proglucagon, a 158 A precursor polypeptide of an amino acid that is processed in different tissues to form several different proglucagon-derived peptides. These proglucagon-derived peptides including, for example, glucagon, GLP-1, glucagon-like peptide-2 (GLP-2), and acidotropin (OXM) are involved in a variety of physiological functions, including blood glucose homeostasis, insulin secretion , gastric emptying and intestinal growth, and regulate food intake.

Therefore, there is a need for therapeutic agents and therapies that mimic the activity of GLP-1 and/or glucagon.

The present invention relates to isolated polypeptides which are glucagon receptor selective analogs and peptide derivatives thereof. Glucagon is a 29-amino acid peptide hormone produced by alpha cells in the pancreas and interacts with the glucagon receptor ("GCGR").

In some embodiments, isolated polypeptides of the present disclosure are glucagon analogs that bind to the glucagon receptor (GCGR) and are selective glucagon receptor agonists. These isolated polypeptides of the present disclosure are potent, stable, and soluble. The isolated polypeptide selectively binds the glucagon receptor relative to native glucagon (eg, human glucagon) and relative to the ability to bind to the GLP-1 receptor. Compared to native glucagon (e.g., human glucagon), isolated peptides exhibit improved metabolic stability and release the drug at rates approaching glomerular filtration rates. The substance is cleared from the kidneys. Isolated polypeptides exhibit improved solubility compared to native glucagon (eg, human glucagon). In preferred embodiments, isolated polypeptides exhibit improved solubility of at least 200 mg/ml. Isolated polypeptides exhibit improved chemical stability at room temperature and at higher temperatures, such as 37°C or higher.

The isolated polypeptides of the present disclosure are derived from a genus of molecules that confer selectivity, solubility and improved self-kidney clearance. This genus is based on (a) identification of key structures required for selectivity at the glucagon receptor, (b) identification of key amino acids and secondary amino acids that provide improved solubility while enhancing or at least maintaining potency of human glucagon. Structural motifs, and (c) identify amino acid substitutions to confer chemical stability to selective glucagon receptor agonists.

In some embodiments, the isolated polypeptides of the present disclosure comprise a modified amino acid sequence based on the human glucagon amino acid sequence: HSQGTFTSDYSKYLDSRRAQDFVQWLMNT-OH (SEQ ID NO: 140), wherein the modified amino acid sequence Including at least one amino acid substitution, at least two amino acid substitutions, at least three amino acid substitutions, at least four amino acid substitutions, at least five amino acid substitutions, at least six amino acid substitutions, at least seven amino acid substitutions amino acid substitution, at least eight amino acid substitutions, at least nine amino acid substitutions, at least 10 amino acid substitutions, at least 11 amino acid substitutions, at least 12 amino acid substitutions, at least 13 amines Amino acid substitution, at least 14 amino acid substitutions, at least 15 amino acid substitutions, at least 16 amino acid substitutions, at least 17 amino acid substitutions, at least 18 amino acid substitutions, at least 19 amino acid substitutions Substitution, at least 20 amino acid substitutions, at least 21 amino acid substitutions, at least 22 amino acid substitutions, at least 23 amino acid substitutions, at least 24 amines Amino acid substitution, at least 25 amino acid substitutions, at least 26 amino acid substitutions, at least 27 amino acid substitutions, at least 28 amino acid substitutions, and/or at least 29 amino acid substitutions, provided that the Isolated polypeptides with modified amino acid sequences retain the ability to act as selective glucagon analogs.

In some embodiments, the isolated polypeptides of the present disclosure comprise a modified amino acid sequence based on the human glucagon amino acid sequence: HSQGTFTSDYSKYLDSRRAQDFVQWLMNT-OH (SEQ ID NO: 140), wherein the modified amino acid sequence Including at least one amino acid substitution, at least two amino acid substitutions, at least three amino acid substitutions, at least four amino acid substitutions, at least five amino acid substitutions, at least six amino acid substitutions, at least seven amino acid substitutions amino acid substitution, at least eight amino acid substitutions, at least nine amino acid substitutions, at least 10 amino acid substitutions, at least 11 amino acid substitutions, at least 12 amino acid substitutions, at least 13 amines amino acid substitution, at least 14 amino acid substitutions, at least 15 amino acid substitutions, or at least 16 amino acid substitutions, wherein the amino acid substitutions are selected from the group consisting of: (i) selected from Y and substituted with an amino acid at position 1 of the group consisting of; (ii) substituted with an amino acid at position 2 selected from the group consisting of G and T; (iii) amine with H at position 3 amino acid substitution; (iv) amino acid substitution of H at position 10; (v) amino acid substitution of T at position 11; (vi) amino acid substitution of R at position 12; (vii) selection Amino acid substitution at position 13 of the group consisting of L and W; (viii) amino acid substitution of E at position 15; (ix) selected from the group consisting of 2-aminoisobutyric acid (Aib), A , amino acid substitution at position 16 of the group consisting of E, I, K, L and Q; (x) amine at position 17 selected from the group consisting of A, E, K, S and T amino acid substitution; (xi) amino acid substitution at position 18 selected from the group consisting of A, E, L and T; (xii) amino acid substitution of E at position 21; (xiii) T at position Amino acid substitution at position 23; (xiv) amino acid substitution at position 24 selected from the group consisting of 2-aminoisobutyric acid (Aib), K and L; (xv) H at position 25 and (xvi) an amino acid substitution of the Z tail at position 30 selected from the group consisting of: EEPSSGAPPPS-OH (SEQ ID NO: 4) EPSSGAPPPS-OH (SEQ ID NO: 5); GAPPPS-OH (SEQ ID NO: 6); GGPSSGAPPPS-OH (SEQ ID NO: 7); GPSSGAPPPS-OH (SEQ ID NO: 8); KRNKNPPPS-OH (SEQ ID NO: 9); KRNKNPPS-OH (SEQ ID NO: 10); KRNKPPIA-OH (SEQ ID NO: 11); KRNKPPPA-OH (SEQ ID NO: 150); KRNKPPPS-OH (SEQ ID NO: 12); KSSGKPPPS-OH (SEQ ID NO: 13 ); PESGAPPPS-OH (SEQ ID NO: 14); PKSGAPPPS-OH (SEQ ID NO: 15); PKSKAPPPS-NH ₂ (SEQ ID NO: 16); PKSKAPPPS-OH (SEQ ID NO: 17); PKSKEPPPS-NH ₂ (SEQ ID NO: 18); PKSKEPPPS-OH (SEQ ID NO: 19); PKSKQPPPS-OH (SEQ ID NO: 20); PSKSKSPPS-NH ₂ (SEQ ID NO: 21); :22); PRNKNNPPS-OH (SEQ ID NO: 23); PSKGAPPPS-OH (SEQ ID NO: 24); PSSGAPPPSE-OH (SEQ ID NO: 25); PSSGAPPPS-NH ₂ (SEQ ID NO: 26); PSSGAPPPS -OH (SEQ ID NO: 27); PSSGAPPPSS-OH (SEQ ID NO: 28); PSSGEPPPS-OH (SEQ ID NO: 29); PSSGKKPPS-OH (SEQ ID NO: 30); PSSGKPPPS-NH ₂ (SEQ ID NO: 31); PSSGKPPPS-OH (SEQ ID NO: 32); PSSGSPPPS-OH (SEQ ID NO: 33); PSSKAPPPS-OH (SEQ ID NO: 34); PSSKEPPPS-OH (SEQ ID NO: 35); PSSKGAPPPS -OH (SEQ ID NO: 36); PSSKQPPPS-OH (SEQ ID NO: 37); PSSKSPPPS-OH (SEQ ID NO: 38); SGAPPPS-OH (SEQ ID NO: 39); and SSGAPPPS-OH (SEQ ID NO: 40); and (xvii) combinations thereof, provided that the isolated polypeptide with the modified amino acid sequence retains the ability to act as a selective glucagon receptor agonist.

In some embodiments, the isolated polypeptide of the present disclosure includes an amino acid sequence selected from the group consisting of the amino acid sequence of the consensus sequence of SEQ ID NO: 1: X ₁ X ₂ X ₃ GTFTSDX ₁₀ X ₁₁ X _{12 X 13 LX 15} X _{16 X 17} X ₁₈ AQEFX _{23 X 24} G or T; X ₃ is Q or H; X ₁₀ is Y or _H ; X ₁₁ is S or T; X ₁₂ is K or R; _{X 13} is Y, L or W; It is S, 2-aminoisobutyric acid (Aib), A, E, L, Q, K or I; X ₁₇ is K, E, S, T or A; X ₁₈ is A, R, S, E, L, T or Y _; X ₂₃ is T or V; X ₂₄ is K, I, L or Aib; SEQ ID NO: 4); EPSSGAPPPS-OH (SEQ ID NO: 5); GAPPPS-OH (SEQ ID NO: 6); GGPSSGAPPPS-OH (SEQ ID NO: 7); GPSSGAPPPS-OH (SEQ ID NO: 8) ; KRNKNPPPS-OH (SEQ ID NO: 9); KRNKNPPS-OH (SEQ ID NO: 10); KRNKPPIA-OH (SEQ ID NO: 11); KRNKPPPA-OH (SEQ ID NO: 150); KRNKPPPS-OH (SEQ ID NO: 12); KSSGKPPPS-OH (SEQ ID NO: 13); PESGAPPPS-OH (SEQ ID NO: 14); PKSGAPPPS-OH (SEQ ID NO: 15); PKSKAPPPS-NH ₂ (SEQ ID NO: 16) ; PKSKAPPPS-OH (SEQ ID NO: 17); PKSKEPPPS-NH ₂ (SEQ ID NO: 18); PKSKEPPPS-OH (SEQ ID NO: 19); PKSKQPPPS-OH (SEQ ID NO: 20); PKSKEPPPS-NH ₂ (SEQ ID NO: 21); PSKSKSPPS-OH (SEQ ID NO: 22); PRNKNNPPS-OH (SEQ ID NO: 23); PSKGAPPPS-OH (SEQ ID NO: 24); PSSGAPPPSE-OH (SEQ ID NO: 25 ); PSSGAPPPS-NH ₂ (SEQ ID NO: 26); PSSGAPPPS-OH (SEQ ID NO: 27); PSSGAPPPSS-OH (SEQ ID NO: 28); PSSGEPPPS-OH (SEQ ID NO: 29); PSSGKKPPS-OH (SEQ ID NO: 30); PSSGKPPPS-NH ₂ (SEQ ID NO: 31); PSSGKPPPS-OH (SEQ ID NO: 32); PSSGSPPPS-OH (SEQ ID NO: 33); PSSKAPPPS-OH (SEQ ID NO: 34); PSSKEPPPPS-OH (SEQ ID NO: 35); PSSKGAPPPS-OH (SEQ ID NO: 36); PSSKQPPPS-OH (SEQ ID NO: 37); PSSKSPPPS-OH (SEQ ID NO: 38); SGAPPPS-OH (SEQ ID NO: 39); and SSGAPPPS-OH (SEQ ID NO: 40).

In some embodiments, the isolated polypeptide of the present disclosure includes an amino acid sequence selected from the group consisting of the amino acid sequence of the consensus sequence of SEQ ID NO: 2: X ₁ X ₂ X ₃ GTFTSDX ₁₀ X ₁₁ X _{12 X 13 LX 15 X 16 _ _ _ _} It is Q or H; X ₁₀ is Y or H; X ₁₁ is S or T; _{X 12} is K or R; X ₁₃ is Y, L or W; X ₁₅ is D or E; Butyric acid (Aib), A or S; X ₁₇ is A or K; X ₁₈ is R, S, L or Y; X ₂₄ is K, I or Aib; X ₂₅ is H or W; and the Z tail does not exist or Selected from the group consisting of: PSSGAPPPS- _NH2 (SEQ ID NO:26); PSSGAPPPS-OH (SEQ ID NO:27); and PSKKSPPPS- _NH2 (SEQ ID NO:21).

In some embodiments, the isolated polypeptide of the present disclosure includes an amino acid sequence selected from the group consisting of the amino acid sequence of the consensus sequence of SEQ ID NO: 3: YSX ₃ GTFTSDYSKYLDX ₁₆ X ₁₇ X ₁₈ AQEFVX ₂₄ WLEDE -Z tail-(OH/NH ₂ ) (SEQ ID NO: 3), where: X ₃ is Q or H; X ₁₆ is 2-aminoisobutyric acid (Aib) or A; X ₁₇ is A or K; _{X 18} is R, _S or Y;

In some embodiments, an isolated polypeptide of the present disclosure includes an amino acid sequence selected from the group consisting of: YSHGTFTSDYSKYLD(Aib)KYAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 41), which is described herein Also known as Compound A1; YSHGTFTSDYSKYLD(Aib)KSAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO:42), which is also known herein as Compound A2; YSQGTFTSDYSKYLDAARAQEFVKWLEDEPKSKSPPPS- _NH2 (SEQ ID NO:43), which is herein Also referred to as Compound A3; YSHGTFTSDYSKYLD(Aib)KRAQEFVIWLEDEPSSGAPPPS- _OH (SEQ ID NO: 44), which is also referred to herein as Compound A4; is Compound A5; and WSQGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 46), which is also referred to herein as Compound A6.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLD(Aib)KYAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 41). In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLD(Aib)KSAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 42). In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSQGTFTSDYSKYLDAARAQEFVKWLEDEPKSKSPPPS-NH ₂ (S EQ ID NO: 43). In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLD(Aib)KRAQEFVIWLEDEPSSGAPPPS-OH (SEQ ID NO: 44). In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLDSARAQEFVKWLEDEPSSGAPPPS- _NH2 (SEQ ID NO: 45). In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence WSQGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 46).

In some embodiments, the isolated polypeptide of the present disclosure consists of the amino acid sequence YSHGTFTSDYSKYLD(Aib)KYAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 41). In some embodiments, the isolated polypeptide of the present disclosure consists of the amino acid sequence YSHGTFTSDYSKYLD(Aib)KSAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 42). In some embodiments, the isolated polypeptide of the present disclosure consists of the amino acid sequence YSQGTFTSDYSKYLDAARAQEFVKWLEDEPKSKSPPPS-NH ₂ (SEQ ID NO: 43). In some embodiments, the isolated polypeptide of the present disclosure consists of the amino acid sequence YSHGTFTSDYSKYLD(Aib)KRAQEFVIWLEDEPSSGAPPPS-OH (SEQ ID NO: 44). In some embodiments, the isolated polypeptide of the present disclosure consists of the amino acid sequence YSHGTFTSDYSKYLDSARAQEFVKWLEDEPSSGAPPPS- _NH2 (SEQ ID NO: 45). In some embodiments, the isolated polypeptide of the present disclosure consists of the amino acid sequence WSQGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 46).

In some embodiments, the isolated polypeptides of the present disclosure comprise selected The amino acid sequence of the group consisting of: YSHGTFTSDYSKYLDAARAQEFVKWLEDEPSSGAPPPS-OH (SEQ ID NO: 143), which is also referred to herein as Compound A97; YSHGTFTSDYTRLLESKRAQEFVKWLEDEPSSGAPPPS-OH (SEQ ID NO: 144), which is also referred to herein as Compound A97 Known as compound A98; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 145), which is also referred to herein as compound A99; 146), which is also referred to herein as Compound A100; YGHGTFTSDHSKYLD(Aib)KRAQEFVKWLEDE-OH (SEQ ID NO: 147), which is also referred to herein as Compound A101; YSHGTFTSDYSKWLD(Aib)KRAQEFVKWLEDE-OH (SEQ ID NO: 148), which is also referred to herein as Compound A102; and YSHGTFTSDYSKYLD(Aib)ARAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 149), which is also referred to herein as Compound A103.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLDAARAQEFVKWLEDEPSSGAPPPS-OH (SEQ ID NO: 143), which is also referred to herein as Compound A97.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYTRLLESKRAQEFVKWLEDEPSSGAPPPS-OH (SEQ ID NO: 144), which is also referred to herein as a compound A98.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 145), which is also referred to herein as Compound A99.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDE-OH (SEQ ID NO: 146), which is also referred to herein as Compound A100.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YGHGTFTSDHSKYLD(Aib)KRAQEFVKWLEDE-OH (SEQ ID NO: 147), which is also referred to herein as Compound A101.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKWLD(Aib)KRAQEFVKWLEDE-OH (SEQ ID NO: 148), which is also referred to herein as Compound A102.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLD(Aib)ARAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 149), which is also referred to herein as Compound A103.

In some embodiments, the polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 143 to 149.

In some embodiments, an isolated polypeptide of the present disclosure includes an amino acid sequence selected from the group consisting of: YSHGTFTSDYSKYLD(Aib)ARAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 47); which is described herein _Also known as Compound A7; NO: 49); which is also referred to herein as Compound A9; YSHGTFTSDYSKYLD (Aib) KRAQEFV (Aib) WLEDEEPSSGAPPPS-OH (SEQ ID NO: 50); which is also referred to herein as Compound A10; YSHGTFTSDYSKYLD (Aib) KRAQEFV ( Aib) WLEDEEEPSSGAPPPS-OH (SEQ ID NO: 51); which is also referred to herein as compound A11; YSHGTFTSDYSKYLD (Aib) KRAQEFV (Aib) WLEDEGAPPPS-OH (SEQ ID NO: 52); which is also referred to herein as compound A12; It is also referred to herein as Compound A14; (SEQ ID NO: 56); which is also referred to herein as Compound A16; YSHGTFTSDYSKYLD (Aib) ERAQEFV (Aib) WLEDEPSSGAPPPS-OH (SEQ ID NO: 57); which is also referred to herein as Compound A17; YSHGTFTSDYSKWLD (Aib )ARAQEFV(Aib)WLEDEPSSGAPPPS-OH(SEQ ID NO:58); which is also referred to herein as Compound A18; YSHGTFTSDYSKWLD(Aib)SRAQEFV(Aib)WLEDEPSSGAPPPS-OH(SEQ ID NO:59); which is also referred to herein as Referred to as Compound A19; 61); which is also referred to herein as Compound A21; YSHGTFTSDYSKYLD (Aib) KRAQEFV (Aib) WLEDEPSSGSPPPS-OH (SEQ ID NO: 62); which is also referred to herein as Compound A22; YSHGTFTSDYSKYLD (Aib) KRAQEFV (Aib) WLEDEPSSKAPPPS-OH (SEQ ID NO: 63); which is also referred to herein as Compound A23; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib) WLEDEPSSKGAPPPS-OH (SEQ ID NO: 64); which is also referred to herein as Compound A24; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGAPPPSS-OH (SEQ ID NO: 65); which is also referred to herein as Compound A25; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGAPPPSE-OH (SEQ ID NO: 66); which is Also referred to herein as Compound A26; ID NO: 68); which is also referred to herein as Compound A28; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPKSGAPPPS-OH (SEQ ID NO: 69); which is also referred to herein as Compound A29; YSHGTFTSDYSKYLD(Aib)KRAQEFV (Aib) WLEDEPSKGAPPPS-OH (SEQ ID NO: 70); which is also referred to herein as Compound A30; YSHGTFTSDYSKYLD (Aib) KRAQEFV (Aib) WLEDEPESGAPPPS-OH (SEQ ID NO: 71); which is also referred to herein as Compound A30; Compound A31; ; which is also referred to herein as Compound A33; YTHGTFTSDHSKWLD(Aib)KRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 74); which is also referred to herein as Compound A34; OH (SEQ ID NO: 75); which is also referred to herein as Compound A35; YSHGTFTSDHSKWLD(Aib)ARAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 76); which is also referred to herein as Compound A36; OH (SEQ ID NO: 77); which is also referred to herein as Compound A37; YSHGTFTSDYSKWLDSARAQEFVKWLEDEPSSGAPPPS-OH (SEQ ID NO: 78); which is also referred to herein as Compound A38; YTHGTFTSDYSKWLDSARAQEFVKWLEDEPSSGAPPPS-OH (SEQ ID NO: 79) ; which is also referred to herein as Compound A39; YSHGTFTSDHSKWLDSARAQEFVKWLEDEPSSGAPPPS-OH (SEQ ID NO: 80); which is also referred to herein as Compound A40; Compound A41; YTHGTFTSDYSKWLDSKRAQEFVKWLEDEPSSGAPPPS-OH (SEQ ID NO: 82); which is also referred to herein as Compound A42; ID NO: 84); which is also referred to herein as Compound A44; YSHGTFTSDYSKYLD (Aib) KRAQEFV (Aib) WLEDEKRNKPPPA-OH (SEQ ID NO: 85); which is also referred to herein as Compound A45; YSHGTFTSDYSKYLD (Aib) KRAQEFV (Aib) WLEDEKRNKPPIA-OH (SEQ ID NO: 86); which is also referred to herein as Compound A46; YSHGTFTSDYSKYLD (Aib) KRAQEFV (Aib) WLEDEKRNKNPPS-OH (SEQ ID NO: 87); which is also referred to herein as Compound A46; Compound A47; ; which is also referred to herein as Compound A49; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEKRNKPPPS-OH (SEQ ID NO: 90); which is also referred to herein as Compound A50; NO: 91); which is also referred to herein as Compound A51; YSHGTFTSDYSKYLDIKRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 92); which is also referred to herein as Compound A52; YSHGTFTSDYSKYLD(Aib)KRAQEFVLWLEDEPSSGAPPPS-OH (SEQ ID NO: 93); which is also referred to herein as Compound A53; YSHGTFTSDYSKYLD (Aib) KRAQEFV (Aib) WLEDEPSSKEPPPPS-OH (SEQ ID NO: 94); which is also referred to herein as Compound A54; YSHGTFTSDYSKYLD (Aib) KRAQEFV ( Aib) WLEDEPSSKSPPPS-OH (SEQ ID NO: 95); which is also referred to herein as Compound A55; YSHGTFTSDYSKYLD (Aib) KRAQEFV (Aib) WLEDEPKSKAPPPS-OH (SEQ ID NO: 96); which is also referred to herein as Compound A55 A56; It is also referred to herein as Compound A58; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSKQPPPS-OH (SEQ ID NO: 99); (SEQ ID NO: 100); which is also referred to herein as Compound A60; YSHGTFTSDYSKYLDSARAQEFVKWLEDEPSSGKPPPS-OH (SEQ ID NO: 101); which is also referred to herein as Compound A61; (SEQ ID NO: 102); which is also referred to herein as Compound A62; YTHGTFTSDYSKYLDSARAQEFVKWLEDEPSSGAPPPS-OH (SEQ ID NO: 103); which is also referred to herein as Compound A63; YTHGTFTSDYSKYLD(Aib)ARAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 104); which _is also referred to herein as Compound A64; Aib)KRAQEFV(Aib)WLEDE PKSKSPPPS-NH ₂ (SEQ ID NO: 106); which is also referred to herein as compound A66; YSHGTFTSDYSKYLD(Aib)ARAQEFV(Aib)WLEDEPKSKSPPPS-NH ₂ (SEQ ID NO: 107); which _Also referred to herein as Compound A67; It is also referred to herein as Compound A69; YSHGTFTSDYSKYLDSARAQEFVKWLEDEPKSKSPPPS-OH (SEQ ID NO: 110); It is also referred to herein as Compound A70; A71; YSHGTFTSDYSKYLDSARAQEFVKWLEDEPKSKEPPPS-NH ₂ (SEQ ID NO: 112); which is also _referred to herein as compound A72; -NH ₂ (SEQ ID NO: 114); which is also referred to herein as Compound A74; WSHGTFTSDYSKYLD (Aib) KRAQEFV (Aib) WLEDEPSSGAPPPS-OH (SEQ ID NO: 115); which is also referred to herein as Compound A75; YSHGTFTSDYSKYLD (Aib )KAAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 116); which is also referred to herein as Compound A76; YSHGTFTSDYSKYLD(Aib)KTAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 117); which is also referred to herein as Referred to as Compound A77; : 119); it is also referred to as compound A79 in this article; YSHGTFTSDYSKYLDAARAQEFVKWLEDEPKSKSPPPS-NH ₂ (SEQ ID NO: 120); it is also referred to as compound A80 in this article; YSQGTFTSDYSKYLDSARAQEFVKWLEDEPKSKSPPPS-OH (SEQ ID NO: 121); Also known as Compound A81; PPPS -OH (SEQ ID NO: 124); which is also referred to herein as Compound A84; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGKPPPS-NH ₂ (SEQ ID NO: 125); which is also referred to herein as Compound A85; YSQGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGKPPPS-OH (SEQ ID NO: 126); which is also referred to herein as compound A86; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEKSSGKPPPS-OH (SEQ ID NO: 127); which is Also referred to herein as Compound A87; ID NO: 129); which is also referred to herein as Compound A8 9; YSHGTFTSDYSKYLD (Aib) KAAQEFV (Aib) WLEDEPSSGKPPPS-OH (SEQ ID NO: 130); which is also referred to herein as Compound A90; WSQGTFTSDYSKYLD (Aib) KRAQEFV(Aib)WLEDEPSSGKPPPS-OH (SEQ ID NO: 131); which is also referred to herein as Compound A91; WSQGTFTSDYSKYLD(Aib)KAAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 132); which is also referred to herein as Compound A91; is Compound A92; WSQGTFTSDYSKYLD(Aib)KAAQEFV(Aib)WLEDEPSSGKPPPS-OH (SEQ ID NO: 133); which is also referred to herein as Compound A93; YSQGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 134) ); which is also referred to herein as Compound A94; YSQGTFTSDYSKYLD(Aib)KAAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 135); which is also referred to herein as Compound A95; and YSQGTFTSDYSKYLD(Aib)KAAQEFV(Aib) WLEDEPSSGKPPPS-OH (SEQ ID NO: 136); which is also referred to herein as Compound A96. In some embodiments, the polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 47 to 136.

In some embodiments, the isolated polypeptide of the present disclosure includes an amino acid sequence selected from the group consisting of the amino acid sequence of the consensus sequence of SEQ ID NO: 137: YSQGTFTSDYSKYLDSX ₁₇ RAQX ₂₁ FVX ₂₄ WLX ₂₇ X ₂₈ T _-OH (SEQ ID NO _: 137) _, where _: K _* is located in the lactam bridge; _{X 24} is K or K** _, where K** is located in the lactam bridge with E or E**, where E** is located in the lactam bridge with K** at X ₂₄ .

In some embodiments, an isolated polypeptide of the present disclosure includes an amino acid sequence selected from the group consisting of: YSQGTFTSDYSKYLDSK*RAQE*FVK**WLDE**T-OH (SEQ ID NO: 138), herein Referred to as Compound A104 and YSQGTFTSDYSKYLDSK*RAQE*FVK**WLQE**T-OH (SEQ ID NO: 139), referred to as Compound A105 herein. In some embodiments, the polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 138 and 139.

Isolated polypeptides of the present disclosure contain amino acid motifs that allow maintenance of characteristics of glucagon receptor-selective analogs (such as solubility and/or stability, such as metabolic stability) compared to native human glucagon molecules, and An amino acid motif that allows maintenance of additional characteristics (such as glucagon selectivity) compared to GLP-1.

In some embodiments, the C-terminal extension of an isolated polypeptide of the present disclosure includes a sequence that binds to serum albumin (eg, human serum albumin). In some embodiments, the C-terminal extension of the isolated polypeptide of the present disclosure is a sequence selected from the group consisting of: EEPSSGAPPPS-OH (SEQ ID NO: 4); EPSSGAPPPS-OH (SEQ ID NO: 5); GAPPPS-OH (SEQ ID NO: 6); GGPSSGAPPPS-OH (SEQ ID NO: 7); GPSSGAPPPS-OH (SEQ ID NO: 8); KRNKNPPPS-OH (SEQ ID NO: 9); KRNKNPPS-OH (SEQ ID NO: 9) NO: 10); KRNKPPIA-OH (SEQ ID NO: 11); KRNKPPPA-OH (SEQ ID NO: 150); KRNKPPPS-OH (SEQ ID NO: 12); KSSGKPPPS-OH (SEQ ID NO: 13); PESGAPPPS -OH (SEQ ID NO: 14); PKSGAPPPS-OH (SEQ ID NO: 15); PKSKAPPPS-NH ₂ (SEQ ID NO: 16); PKSKAPPPS-OH (SEQ ID NO: 17); PKSKEPPPS-NH ₂ (SEQ ID NO: 18); PKSKEPPPS-OH (SEQ ID NO: 19); PKSKQPPPS-OH (SEQ ID NO: 20); PKSKEPPPS-NH ₂ (SEQ ID NO: 21); PSKSKSPPS-OH (SEQ ID NO: 22) ; PRNKNNPPS-OH (SEQ ID NO: 23); PSKGAPPPS-OH (SEQ ID NO: 24); PSSGAPPPSE-OH (SEQ ID NO: 25); PSSGAPPPS-NH ₂ (SEQ ID NO: 26); PSSGAPPPS-OH ( SEQ ID NO: 27); PSSGAPPPSS-OH (SEQ ID NO: 28); PSSGEPPPS-OH (SEQ ID NO: 29); PSSGKKPPS-OH (SEQ ID NO: 30); PSSGKPPPS-NH ₂ (SEQ ID NO: 31 ); PSSGKPPPS-OH (SEQ ID NO: 32); PSSGSPPPS-OH (SEQ ID NO: 33); PSSKAPPPS-OH (SEQ ID NO: 34); PSSKEPPPS-OH (SEQ ID NO: 35); PSSKGAPPPS-OH ( SEQ ID NO: 36); PSSKQPPPS-OH (SEQ ID NO: 37); PSSKSPPPS-OH (SEQ ID NO: 38); SGAPPPS-OH (SEQ ID NO: 39); and SSGAPPPS-OH (SEQ ID NO: 40 ).

In some embodiments, the C-terminal amine of the isolated polypeptide of the present disclosure The carboxyl group of the amino acid residue is acylaminated. In some embodiments, the carboxyl group of the C-terminal amino acid residue of the isolated polypeptide of the present disclosure is unmodified.

In some embodiments, the isolated polypeptides provided herein are glucagon active agonists. In some embodiments, the isolated polypeptides provided herein can bind to the glucagon receptor. In some embodiments, the glucagon receptor is a human glucagon receptor. In some embodiments, an isolated polypeptide of the present disclosure has a pEC50 in a range of greater than about 9.0 with human liters in a cAMP assay (as described herein) using an 11-point curve starting in the range of 1 nM to 500 micromolar. Glucose receptor binding. In some embodiments, an isolated polypeptide of the present disclosure exhibits a pEC50 in a range greater than about 11.0 in a cAMP assay (as described herein) using an 11-point curve starting in the range of 1 nM to 500 micromolar with human liters Glucose receptor binding.

In some embodiments, isolated polypeptides of the present disclosure bind to the human glucagon receptor but do not substantially bind to the human GLP-1 receptor. As used herein, the term "substantially does not bind" and variations thereof refer to polypeptides that exhibit low to no affinity for the human GLP-1 receptor. In some embodiments, an isolated polypeptide of the present disclosure binds to the human glucagon receptor with an affinity that is at least 100-fold greater than the affinity of the same isolated polypeptide to the human GLP-1 receptor. In preferred embodiments, an isolated polypeptide of the present disclosure binds to the human glucagon receptor with an affinity that is at least 1,000 times greater than the affinity of the same isolated polypeptide to the human GLP-1 receptor. In some embodiments, an isolated polypeptide of the present disclosure binds to the human glucagon receptor with a pEC50 in a range greater than about 9.0 in a cAMP assay using an 11-point curve starting in the range of 1 nM to 500 micromolar, and the present invention Isolated polypeptides disclosed have a cAMP assay of less than about 10.0 The pEC50 binds to the human GLP-1 receptor. In some embodiments, an isolated polypeptide of the present disclosure binds to the human glucagon receptor with a pEC50 in a range greater than about 11.0 in a cAMP assay using an 11-point curve starting in the range of 1 nM to 500 micromolar, and the present invention The isolated polypeptides disclosed bind to the human GLP-1 receptor with a pEC50 of less than about 9.0 in a cAMP assay.

In some embodiments, an isolated polypeptide provided herein can further comprise a heterologous moiety associated with the polypeptide. In some embodiments, the heterologous moiety is a protein, peptide, protein domain, linker, organic polymer, inorganic polymer, polyethylene glycol (PEG), biotin, albumin, human serum albumin ( HSA), HSA FcRn binding portion, antibody, antibody domain, antibody fragment, single chain antibody, domain antibody, albumin binding domain, enzyme, ligand, receptor, binding peptide, non-FnIII scaffold, epitope tag , recombinant polypeptide polymers, interleukins, or any combination of two or more of these parts.

Isolated polypeptides provided herein exhibit glucagon receptor agonist activity, for example, by binding to the glucagon receptor. When binding to or otherwise interacting with a glucagon receptor, the isolated polypeptides provided herein fully or partially agonize or otherwise stimulate glucagon activity. Stimulation or modulation of the biological function of glucagon depends, in whole or in part, on the interaction between a glucagon receptor agonist and the glucagon receptor.

The isolated polypeptides of the present disclosure are selective glucagon receptor agonists and can be used alone or in combination with at least a second agent. In preferred embodiments, the second agent is a polypeptide. In preferred embodiments, the second polypeptide is an insulinotropic peptide. For example, the insulin-stimulating polypeptide peptide is selected from exenatide, exenatide, A group consisting of peptide derivatives, exenatide analogs, glucagon-like peptide-1 (GLP-1), GLP-1 derivatives and GLP-1 analogs.

In preferred embodiments, the insulinotropic polypeptide is exenatide, an exenatide derivative or an exenatide analog. In preferred embodiments, exenatide is synthetic exenatide. In a preferred embodiment, the synthetic exenatide contains the amino acid sequence H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu- Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ (SEQ ID NO: 142).

In combination therapy, the use of a selective glucagon analog allows titration to find the appropriate therapeutic dose of glucagon and any GLP-1 receptor agonist. This allows for the desired effects of glucagon/GLP-1 agonism (ie, weight loss, increased energy expenditure) but without harmful blood glucose spikes.

In some embodiments, the isolated polypeptides and additional agents provided herein are formulated into a single therapeutic composition, and the isolated polypeptide and additional agents are administered simultaneously. In some embodiments, the isolated polypeptide and the additional agent are separated from each other, e.g., each is formulated into separate therapeutic compositions and the isolated polypeptide and the additional agent are administered simultaneously, or the isolated polypeptide and the additional agent are administered in a treatment regimen administered at different times during the period. For example, the isolated polypeptide is administered before the additional dose is administered, the isolated polypeptide is administered after the additional dose is administered, or the isolated polypeptide and the additional dose are administered in an alternating manner. As described herein, isolated polypeptides and additional agents are administered in a single dose or in multiple doses.

Also provided herein are treatments to delay the onset, delay the progression, or otherwise ameliorate abnormalities characterized by abnormal glucagon activity. Methods for treating symptoms of diseases or conditions that are associated with normal glucagon activity, or are otherwise associated with abnormal glucagon activity. In some embodiments, the disease or condition is type 2 diabetes.

Also provided herein are methods of treating metabolic disorders by administering to an individual in need thereof an isolated polypeptide of the present disclosure or any pharmaceutical composition described herein.

Also provided herein are methods of treating obesity by administering to an individual in need thereof an isolated polypeptide of the present disclosure or any pharmaceutical composition described herein.

Also provided herein are methods of treating, preventing, delaying the onset, delaying progression, and/or otherwise ameliorating the symptoms of metabolic diseases or disorders associated with elevated blood sugar in a patient by administering the drug to the patient in need thereof. to the isolated polypeptides of the present disclosure or any pharmaceutical composition described herein.

Also provided herein are methods of treating, preventing, delaying the onset, delaying progression, and/or otherwise ameliorating symptoms of a disease or disorder in which agonistic glucagon receptor action is desired, such as the following Non-limiting examples: chronic pain, hemophilia and other blood disorders, endocrinology, metabolic disorders, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), Alzheimer's disease, cardiovascular disease Disease, asymptomatic hypoglycemia, restrictive pulmonary disease, chronic obstructive pulmonary disease, lipoatrophy, metabolic syndrome, leukemia, hepatitis, renal failure, autoimmune diseases (e.g., Grave's disease, erythema systemicis) Lupus, multiple sclerosis and rheumatoid arthritis), shock and/or wasting disease, pancreatitis, and neurological disorders and diseases such as Parkinson's disease.

Also provided herein are methods of treating, preventing, delaying the onset, delaying progression, and/or otherwise ameliorating symptoms of infectious diseases requiring chronic treatment.

In the methods disclosed herein, the isolated polypeptides of the present disclosure and/or the pharmaceutical compositions described herein are administered in single or multiple doses alone or with pharmaceutically acceptable carriers and/or excipients and/or Or administered in combination with polymers and/or organic solvents.

Pharmaceutical compositions according to the present invention may include the polypeptides of the present disclosure together with suitable carriers. These pharmaceutical compositions may be included in kits, such as, for example, diagnostic kits.

Figures 1A and 1B are a series of graphs depicting the effects of human glucagon, GLP-1(7-37), and two glucagon receptor-selective peptide agonists on human glucagon receptor (GCGR) and human GLP- Peptide-receptor activation profile of 1 receptor (GLP-1R). Figure 1A shows that the peptides referred to herein as Compound A2 and Compound Al and glucagon are nearly equally potent on GCGR, whereas GLP-1 (7-37) activates GCGR with much less potency. Figure 1B shows that compared to the results in Figure 1A, peptide compound A2 and compound A1 are inactive towards GLP-1R, indicating that peptide compound A2 and compound A1 function as GCGR selective agonists.

Figures 2A and 2B are a series of graphs and a table depicting the mean plasma concentration versus time of various glucagon analogues following a 3-hr intravenous infusion in male rats at a dose of 0.3 mg/kg. Figure 2A depicts compounds referred to herein as Compound A99 (SEQ ID NO: 145), Compound A102 (SEQ ID NO: 148), Glucagon analogs of compound A98 (SEQ ID NO: 144), compound A100 (SEQ ID NO: 146), and compound A101 (SEQ ID NO: 147), where n=3 to 6, and the error bars in the figure represents the standard error, and the CL values in the table are shown as the mean ± standard error (n=3 to 6). Figure 2B depicts glucagon analogs referred to herein as Compound A2, Compound A1, Compound A5, Compound A6, Compound A4, and Compound A3, where n=3 to 5, and the error bars in the figure represent standard errors, And the CL values in the table are shown as the mean ± standard error (n=3 to 5).

Figures 3A, 3B, and 3C are a series of graphs depicting the efficacy of various glucagon analogues, measuring body weight changes in DIO rats after 13 days. Specifically, Figure 3A depicts the efficacy of a glucagon analog, referred to herein as Compound A104, when administered alone (and alone) or in combination with exendin-4. Figure 3B depicts the efficacy of a glucagon analog, referred to herein as Compound A2, and Figure 3C depicts the efficacy of a glucagon analog, referred to herein as Compound Al. In all graphs, p<0.05 compared to vehicle control.

Figures 4A and 4B are schematic diagrams of the chemical structures of cyclic peptide glucagon analogs referred to herein as Compound A104 (Figure 4A) and Compound A105 (Figure 4B).

Figure 5 plots steady-state plasma concentrations of exenatide (circles) and GLP-1 (triangles) on the Y-axis against intravenous infusion rate on the X-axis, and compares these correlations with those of inulin (which is Cleared by glomerular filtration). The data in Figure 5 shows that the steady-state plasma concentration of exenatide is close to that of inulin, and exenatide is mainly transported through the glomerulus. Filter clear consistency.

Figure 6 shows the steady state of glucagon (inverted triangle) and compounds A1 and A2 (glucagon analogs, squares) in addition to exenatide (circles) and GLP-1 (triangles) in Figure 5 Plasma concentration is plotted on the Y-axis against intravenous infusion rate on the X-axis. These correlations are shown relative to inulin (estimate of glomerular filtration rate). The data in Figure 6 show that the steady-state plasma concentrations of compounds A1 and A2 are close to those of inulin, consistent with the fact that these compounds are primarily cleared by glomerular filtration.

Figure 7 shows the clearance values for glucagon, compounds A1, A2 and A3 (the three points closest to the GFR) and other glucagon analogs. Note that the washout value decreases along the y-axis. For comparison, the upper dashed line (closer to the GFR) shows the exenatide clearance rate and the lower dashed line (further from the GFR) shows the glucagon clearance rate. Figure 7 shows that the clearance of certain glucagon analogs, including compounds A1, A2 and A3, is less than that of exenatide and glucagon.

Figure 8 is a graph comparing dose-dependent weight loss in male Sprague-Dawley rats following subcutaneous administration of exenatide or Compound Al.

Figure 9 is a graph comparing dose-dependent weight loss in male Sprague-Dawley rats following subcutaneous administration of Compound A1, A2 or A3.

Figure 10 is a graph comparing the body weight of DIO LE rats after 27 days of administration of various doses of exenatide, a glucagon ("GCG") analog, Compound A1, or a combination of exenatide and a GCG analog. alleviate.

Figure 11 is a bar graph alternatively depicting the data shown in Figure 10.

Figure 12 is a graph comparing serum triglyceride levels in Zucker diabetic fatty liver (ZDF) rats 14 days after administration of Compound A104 (alone or in combination with exenatide).

Figure 13 is a graph comparing serum liver fat content and liver weight in Zucker diabetic fatty liver (ZDF) rats administered different doses of Compound A104 for 14 days. Note that liver weight is plotted on the left Y-axis as an open circle. Liver fat is plotted as a solid triangle on the right Y-axis.

Figure 14 shows a 3-D graph showing weight loss in DIO LE rats caused by continuous administration of exenatide or glucagon analogues.

Figure 15 depicts 3-D assessment of body weight loss in DIO LE rats following continuous administration of the exenatide/glucagon analog combination.

Figure 16 depicts a 3-D assessment of body weight loss in DIO LE rats following continuous administration of a fixed ratio exenatide/glucagon analog combination.

Figure 17 is a graphical evaluation of the in vivo potency of exenatide/glucagon analog combinations at different fixed ratios. In vivo potency is defined as the effect per unit of total drug mass.

The present invention relates to isolated polypeptides which are glucagon receptor selective analogs and peptide derivatives thereof. Glucagon is produced by the pancreas and interacts with the glucagon receptor ("GCGR"). In some embodiments, isolated polypeptides of the present disclosure are selective glucagon receptor agonists. In some embodiments, isolated polypeptides of the present disclosure bind to glucagon receptors.

definition

It should be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting. As used in this specification and the accompanying claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a solvent" includes a combination of two or more such solvents, reference to "a peptide" includes one or more peptides or a mixture of peptides, reference to "a drug" "a drug" includes one or more drugs, and "an osmotic delivery device" includes one or more osmotic delivery devices and the like. Unless specifically stated otherwise or obvious from the context, the term "or" as used herein shall be understood to be inclusive and cover both "or" and "and".

Unless specifically stated or obvious from the context, the term "about" used herein should be understood to mean within the normal allowable range in the art, such as within 2 standard deviations of the mean. Approximately can be understood as 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the stated value Within. Unless clear from the context, all numerical values provided herein are qualified by the word "about."

Unless specifically stated or obvious from the context, the term "substantially" used herein shall be understood to mean within a narrow range of variation or other normal allowable values in the art. Substantially can be understood as being within 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or 0.001% of the stated value.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although other methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein.

In describing and claiming the present invention, the following terms will be used according to the definitions set forth below.

The terms "drug," "therapeutic agent" and "beneficial agent" are used interchangeably to refer to any therapeutically active substance that is delivered to an individual to produce a desired beneficial effect. In one embodiment of the invention, the drug is a polypeptide. In another embodiment of the invention, the drug is a small molecule, such as a hormone such as androgen or estrogen. The devices and methods of the present invention are well suited for the delivery of proteins, small molecules, and combinations thereof.

The terms "peptide", "polypeptide" and "protein" are used interchangeably herein and generally refer to a compound containing two or more amino acids (e.g., most generally L -Amino acids, but also include, for example, molecules of chains of D-amino acids, modified amino acids, amino acid analogs and amino acid mimetics). Peptides can be naturally occurring, synthetically produced, or recombinantly expressed. Peptides may also contain additional groups that modify the amino acid chain, such as adding functional groups via post-translational modifications. Examples of post-translational modifications include, but are not limited to, acetylation, alkylation (including methylation), biotinylation, glutamylation, glycylation, glycosylation, isoprene formation, lipidation, phosphorylation, selenization and C-terminal amidation. The term peptide also includes peptides containing amino-terminal and/or carboxyl-terminal modifications. Modifications of the terminal amine groups include, but are not limited to, deamination, N-lower alkyl, N-di-lower alkyl, and N-carboxyl modifications. Modifications of the terminal carboxyl groups include, but are not limited to, amide, lower alkylamide, dialkylamide and lower alkyl ester modifications (for example, where the lower alkyl group is a C ₁ -C ₄ alkyl group). The term peptide also includes modifications of the amino acid between the amine terminus and the carboxyl terminus, such as, but not limited to, those described above. In one embodiment, peptides can be modified by adding small molecule drugs.

The terminal amino acid at one end of the peptide chain generally has a free amine group (i.e., the amine terminus). The terminal amino acid at the other end of the peptide chain generally has a free carboxyl group (i.e., the carboxyl terminus). Generally speaking, the amino acids that make up a peptide are numbered sequentially, starting from the amino end of the peptide toward the carboxyl end.

The term "amino acid residue" as used herein refers to an amino acid incorporated into a peptide through a amide bond or a amide bond mimetic.

The term "insulinotrophic" as used herein generally refers to the ability of a compound (eg, a peptide) to stimulate or affect the production and/or activity of insulin (eg, an insulinotropic hormone). The compound generally stimulates or otherwise affects the secretion or production of insulin in an individual. Therefore, "insulinotrophic peptide" is an amino acid-containing molecule that can stimulate or otherwise affect insulin secretion or synthesis.

The term "insulinotrophic peptide" as used herein includes, but is not limited to, glucagon-like peptides 1 (GLP-1) and its derivatives and analogs, exenatide, exenatide having the amino acid sequence of SEQ ID NO: 1, and its derivatives and analogs.

The term "incretin mimetics" used herein includes, but is not limited to, GLP-1 peptides, peptide derivatives of GLP-1, and peptide analogs of GLP-1; exenatide, having SEQ. The amino acid sequence of ID NO: 1 is exenatide, exenatide peptide, peptide derivatives of exenatide and peptide analogs of exenatide. Examples of preferred incretin mimetics include exenatide, an amino acid having secretin-4 (the naturally occurring form of exenatide and having the amino acid sequence of SEQ ID NO: 1) Sequence of exenatide, exenatide-LAR, lixisenatide, GLP-1(7-36), liraglutide, semaglutide, dulaglutide peptide (dulaglutide), albiglutide (albiglutide), and taspoglutide (taspoglutide). Incretin mimetics are also referred to herein as "insulin-stimulating peptides." Incretin mimetics that target the GLP-1 receptor are also called "GLP-1 receptor agonists" in the literature.

The term "an exenatide" used herein includes, but is not limited to, exenatide, exenatide having the amino acid sequence of SEQ ID NO: 1, natural insulin secretin-4, Exenatide peptide, exenatide analogs and exenatide derivatives.

The term "GLP-1" refers to a polypeptide produced by L-cells located primarily in the ileum and colon, and to a lesser extent in the duodenum and jejunum. GLP-1 is a regulatory peptide of the G-coupled protein receptor on β cells. It stimulates the intestinal tract through the activity of adenylyl cyclase and the production of cAMP. Insulin response to absorbed nutrients [Baggio 2007, "Biology of incretins: GLP-1 and GIP," Gastroenterology, vol. 132(6): 2131-57; Holst 2008, "The incretin system and its role in type 2 diabetes mellitus ," Mol Cell Endocrinology, vol. 297(1-2): 127-36]. The effects of GLP-1R agonism are multiple. GLP-1 enhances endogenous glucose-dependent insulin secretion, makes β-cells glucose competent and sensitive to GLP-1, inhibits glucagon release, restores first and second phase insulin secretion, and delays gastric Empty, reduce food intake, and increase satiety to maintain blood glucose stability [Holst 2008 Mol. Cell Endocrinology; Kjems 2003 "The influence of GLP-1 on glucose-stimulated insulin secretion: effects on beta-cell sensitivity in type 2 and nondiabetic subjects," Diabetes, vol. 52(2): 380-86; Holst 2013 "Incretin hormones and the satiation signal," Int J Obes(Lond), vol. 37(9): 1161-69; Seufert 2014," The extra-pancreatic effects of GLP-1 receptor agonists: a focus on the cardiovascular, gastrointestinal and central nervous systems,” Diabetes Obes Metab, vol. 16(8): 673-88]. Considering the mode of action of GLP-1, the risk of hypoglycemia is minimal.

The term "glucagon" refers to a 29-amino acid peptide hormone produced by alpha cells in the pancreas and which interacts with GCGR. The amino acid sequence of glucagon is HSQGTFTSDYSKYLDSRRAQDFVQWLMNT-OH (SEQ ID NO: 140).

As used herein, the term "glucagon analog" means a structure similar to glucagon and the term "glucagon receptor agonist" describes the function of a glucagon receptor. Compounds that act as agonists for the body. The terms "glucagon analog" and "glucagon receptor agonist" are used interchangeably to describe the peptides disclosed herein.

Glucagon is derived from the 158-amino acid pro-proglucagon peptide, which also functions as the peptide hormones GLP-1, GLP-2, glucagon, glicentin, and glucagon-related Precursor of tissue-specific processing of pancreatic polypeptide. Glucagon corresponds to amino acid residues 33 to 61 of the precursor peptide and acts via interaction with type B 7-transmembrane G protein-coupled receptors located primarily in the liver. Immunostaining also demonstrated the presence of glucagon receptors in the kidneys, gastrointestinal tract, heart, spleen, brain, adipocytes, and lymphoblastoid cells. Glucagon is released in response to low blood sugar concentrations and stimulates glycogen output via glycolysis and glucose production. Glucagon acts as a countermeasure to the hypoglycemic effect of insulin and is tightly controlled via a feedback system between the two hormones to allow effective blood glucose homeostasis.

In addition to its effects on blood glucose concentrations, glucagon also increases energy expenditure and thermogenesis. Increased signaling has a direct effect on the regulation of triglycerides, free fatty acids, apolipoproteins, and bile acid metabolism. The current therapeutic use of glucagon is mainly focused on its use as a rescue agent when blood sugar is too low. However, recent studies have exploited the ability of hormones to influence energy balance and lipid metabolism, leading to potential treatments for a variety of metabolic disorders.

The low solubility of glucagon (<1.0 mg/ml in aqueous or saline buffer) precludes its use in chronic trials administered via continuous infusion. The solubility of glucagon and the pEC50 values determined in the cAMP assay for glucagon receptor (GLUR) and GLP1 are shown in Table 1 below:

The term "vehicle" as used herein refers to a medium used to carry a compound (eg, a drug or drug-containing particles). Vehicles of the present invention generally include components such as polymers and solvents. Suspension vehicles of the present invention generally include solvents and polymers used to prepare suspension formulations that further include drug particle formulations.

The term "phase separation" as used herein refers to the formation of multiple phases (eg, liquid and colloidal phases) in a suspending vehicle, such as when the suspending vehicle comes into contact with an aqueous environment. In some embodiments of the invention, the suspending vehicle is formulated to exhibit phase separation when exposed to an aqueous environment having less than about 10% water.

The term "single-phase" as used herein refers to a solid, semi-solid or liquid homogeneous system that is physically and chemically uniform throughout.

The term "dispersed" as used herein means dissolving, dispersing, suspending or otherwise distributing a compound (eg, a pharmaceutical particulate formulation) in a suspending vehicle.

The term "chemically stable" as used herein refers to the formation of an acceptable percentage of degradation products in a formulation over a defined period of time through chemical pathways such as deamidation (usually by hydrolysis), produced by aggregation or oxidation.

The term "physically stable" as used herein refers to the formation of an acceptable percentage of aggregates (eg, dimers and other higher molecular weight products) in the formulation. Additionally, physically stable formulations do not change their physical state, such as from liquid to solid, or from amorphous to crystalline form.

The term "viscosity" as used herein generally refers to a value determined from the ratio of shear stress to shear rate (see, e.g., Considine, D.M. & Considine, G.D., Encyclopedia of Chemistry, 4th Edition, Van Nostrand, Reinhold ,N.Y.,1984), basically as follows: F/A=μ*V/L (Equation 1)

Where F/A=shear stress (force per unit area), μ=proportionality constant (viscosity), and V/L=flow rate per layer thickness (shear rate).

From this relationship, the ratio of shear stress to shear rate defines viscosity. Measurement of shear stress and shear rate is generally determined using a parallel plate rheometer under selected conditions (eg, a temperature of about 37°C). Other methods for determining viscosity include measuring dynamic viscosity using a viscometer, such as the Cannon-Fenske Viscometer, the Ubbelohde Viscometer for Cannon-Fenske Opaque Solutions, or the Ostwald Viscometer. Typically, the suspension of the present invention The vehicle has a viscosity sufficient to prevent the particulate formulation suspended therein from settling during storage and use in a delivery method, such as in an implantable drug delivery device.

The term "non-aqueous" as used herein means, for example, that the overall water content of the suspension formulation is generally less than or equal to about 10 wt%, such as less than or equal to about 7 wt%, less than or equal to about 5 wt%, and/or or less than about 4 wt%. Likewise, the granular formulations of the present invention contain a residual moisture content of less than about 10 wt%, such as less than about 5 wt%.

The term "subject" as used herein refers to any member of the subphylum Chordata, including but not limited to humans and other primates, including non-human primates such as rhesus macaques and other monkey species and chimpanzees and other ape species; farm animals such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats, and guinea pigs; birds, including domestic poultry, wild birds, and game birds Birds such as chickens, pheasants and other quail birds, ducks, geese and the like are used. The term does not imply a specific age or gender. Therefore, both adult and newborn individuals are covered.

The term "osmotic delivery device" as used herein generally refers to a device for delivering a drug (eg, an isolated glucagon-specific agonist polypeptide) to an individual, wherein the device includes, for example, a chamber A storage tank (made of, for example, titanium alloy), the chamber contains a suspension formulation and an osmotic agent formulation containing a drug (eg, an isolated glucagon-specific agonist polypeptide). The single suspension formula of the piston assembly located in the chamber and Penetrant formulation. A semipermeable membrane is located at a first distal end adjacent the reservoir of the penetrant formulation and a diffusion modifier (which defines a delivery aperture through which the suspended formulation exits the device) is located at a second distal end adjacent the reservoir of the suspended formulation. Generally, osmotic delivery devices are implanted within an individual, such as subdermally or subcutaneously (eg, within, outside, or behind the upper arm and abdominal areas). An exemplary osmotic delivery device is the DUROS® (ALZA Corporation, Mountain View, Calif.) delivery device. Examples of terms synonymous with "osmotic delivery device" include, but are not limited to, "osmotic drug delivery device", "osmotic drug delivery system", "osmotic device", "osmotic delivery device", "osmotic delivery system", "osmotic pump", "Implantable drug delivery device", "drug delivery system", "drug delivery device", "implantable osmotic pump", "implantable drug delivery system" and "implantable delivery system". Other terms for "osmotic delivery devices" are known in the art.

The term "continuous delivery" as used herein generally refers to the substantially continuous release of drug from an osmotic delivery device into tissues close to the implantation site, such as subdermis and subcutaneous tissue. For example, osmotic delivery devices release drugs at a predetermined rate based essentially on the principle of osmosis. Extracellular fluid passes directly through the semipermeable membrane into the osmotic engine of the osmotic delivery device, which expands at a slow and consistent stroke rate to drive the piston. The piston movement forces the release of the drug formula through the holes of the diffusion regulator. Drug release from the osmotic delivery device therefore occurs at a slow, controlled, and consistent rate.

The term "substantial steady-state delivery" as used herein generally refers to a defined The drug is delivered at or near a target concentration during the interval, wherein the amount of drug delivered from the osmotic delivery device is substantially zero-order delivery. Substantially zero-order delivery of an active agent (e.g., an isolated glucagon-specific analogue) means that the rate of drug delivery is constant and independent of the drug available in the delivery system; for example, with respect to zero-order delivery, if the drug is delivered The rate is plotted against time and a line fitted to the data is obtained, with a slope that is approximately zero as determined by standard methods (such as linear regression).

The term "drug half-life" as used herein refers to the length of time required for a drug to clear half of its concentration from the blood plasma. The half-life of a drug is usually measured by monitoring how the drug degrades when administered by injection or intravenously. Drugs are typically detected using, for example, radioimmunoassay (RIA), chromatography, electrochemiluminescence (ECL) assay, enzyme immunosorbent assay (ELISA) or immunoenzyme sandwich assay (IEMA).

The terms "μg" and "mcg" and "ug" should be understood to mean "micrograms". Similarly, the terms "μl" and "uL" should be understood to mean "microliter", and the terms "μM" and "uM" should be understood to mean "micromolar".

The term "serum" means any blood preparation in which substances can be detected. The term serum therefore includes at least whole blood, serum and plasma. For example, "the amount of [substance] in the serum of an individual" would encompass "the amount of [substance] in the plasma of an individual."

The term "LOCF" means "Last Observation Carried Forward". LOCF is an imputation method that can be used to analyze longitudinal clinical trial data to deal with dropouts. exist In the LOCF method, the last observed non-missed value is used to fill in the missed values at later time points. As used herein, LOCF includes the last post-randomization data obtained from individuals who had at least one post-randomization HbA1c value when they discontinued participation in the trial.

The term "mITT" stands for "Modified Intention-to-Treat Population." In this article, the primary efficacy and safety analyzes will be conducted in the mITT population, which includes all randomly assigned individuals who receive at least one ITCA 650 or ITCA placebo treatment, have a valid baseline, and have at least one post-baseline HbA1c value.

Baseline was defined as the last assessment on or before the day of initial placement of the ITCA 650 osmotic delivery device (containing drug or placebo).

2.0.0 Overview of the invention

Before the present invention is described in detail, it is to be understood that this invention is not limited to specific types of drug delivery devices, specific sources of drugs, specific solvents, specific polymers, and the like, as the use of such specifics can be selected in light of the teachings of this specification. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

In a first aspect, the invention relates to isolated polypeptides which are glucagon receptor selective analogs and peptide derivatives thereof. In some embodiments, isolated polypeptides of the present disclosure are selective glucagon receptor agonists. In some embodiments, isolated polypeptides of the disclosure bind to the glucagon receptor (GCGR).

In some embodiments, the isolated polypeptides of the present disclosure comprise a modified amino acid sequence based on the human glucagon amino acid sequence: HSQGTFTSDYSKYLDSRRAQDFVQWLMNT-OH (SEQ ID NO: 140), wherein the modified amino acid sequence Including at least one amino acid substitution, at least two amino acid substitutions, at least three amino acid substitutions, at least four amino acid substitutions, at least five amino acid substitutions, at least six amino acid substitutions, at least seven amino acid substitutions amino acid substitution, at least eight amino acid substitutions, at least nine amino acid substitutions, at least 10 amino acid substitutions, at least 11 amino acid substitutions, at least 12 amino acid substitutions, at least 13 amines Amino acid substitution, at least 14 amino acid substitutions, at least 15 amino acid substitutions, at least 16 amino acid substitutions, at least 17 amino acid substitutions, at least 18 amino acid substitutions, at least 19 amino acid substitutions Substitution, at least 20 amino acid substitutions, at least 21 amino acid substitutions, at least 22 amino acid substitutions, at least 23 amino acid substitutions, at least 24 amino acid substitutions, at least 25 amino acid substitutions, At least 26 amino acid substitutions, at least 27 amino acid substitutions, at least 28 amino acid substitutions, and/or at least 29 amino acid substitutions, provided that the isolated polypeptide with the modified amino acid sequence remains Ability to act as a selective glucagon analogue.

In some embodiments, the isolated polypeptides of the present disclosure comprise a modified amino acid sequence based on the human glucagon amino acid sequence: HSQGTFTSDYSKYLDSRRAQDFVQWLMNT-OH (SEQ ID NO: 140), wherein the modified amino acid sequence Including at least one amino acid substitution, at least two amino acid substitutions, at least three amino acid substitutions, at least four amino acid substitutions, at least five amino acid substitutions, at least six amino acid substitutions, at least seven amino acid substitutions amino acid substitution, at least eight amino acid substitutions, at least nine amino acid substitutions, at least 10 amino acid substitutions, at least 11 amino acid substitutions, at least 12 amino acid substitutions, at least 13 amines amino acid substitution, at least 14 amino acid substitutions, at least 15 amino acid substitutions, or at least 16 amino acid substitutions, wherein the amino acid substitutions are selected from the group consisting of: (i) selected from Y and substituted with an amino acid at position 1 of the group consisting of; (ii) substituted with an amino acid at position 2 selected from the group consisting of G and T; (iii) amine with H at position 3 amino acid substitution; (iv) amino acid substitution of H at position 10; (v) amino acid substitution of T at position 11; (vi) amino acid substitution of R at position 12; (vii) selection Amino acid substitution at position 13 of the group consisting of L and W; (viii) amino acid substitution of E at position 15; (ix) selected from the group consisting of 2-aminoisobutyric acid (Aib), A , amino acid substitution at position 16 of the group consisting of E, I, K, L and Q; (x) amine at position 17 selected from the group consisting of A, E, K, S and T amino acid substitution; (xi) amino acid substitution at position 18 selected from the group consisting of A, E, L and T; (xii) amino acid substitution of E at position 21; (xiii) T at position 21 Amino acid substitution at position 23; (xiv) amino acid substitution at position 24 selected from the group consisting of 2-aminoisobutyric acid (Aib), K and L; (xv) H at position 25 amino acid substitution at position; (xvi) amino acid substitution at position 30 of the Z tail selected from the group consisting of: EEPSSGAPPPS-OH (SEQ ID NO: 4) EPSSGAPPPS-OH (SEQ ID NO: 5 ); GAPPPS-OH (SEQ ID NO: 6); GGPSSGAPPPS-OH (SEQ ID NO: 7); GPSSGAPPPS-OH (SEQ ID NO: 8); KRNKNPPPS-OH (SEQ ID NO: 9); KRNKNPPS-OH ( SEQ ID NO: 10); KRNKPPIA-OH (SEQ ID NO: 11); KRNKPPPA-OH (SEQ ID NO: 150); KRNKPPPS-OH (SEQ ID NO: 12); KSSGKPPPS-OH (SEQ ID NO: 13) ; PESGAPPPS-OH (SEQ ID NO: 14); PKSGAPPPS-OH (SEQ ID NO: 15); PKSKAPPPS-NH ₂ (SEQ ID NO: 16); PKSKAPPPS-OH (SEQ ID NO: 17); PKSKEPPPS-NH ₂ (SEQ ID NO: 18); PKSKEPPPS-OH (SEQ ID NO: 19); PKSKQPPPS-OH (SEQ ID NO: 20); PSKSKSPPS-NH ₂ (SEQ ID NO: 21); PSKSKPPPS-OH (SEQ ID NO: 22); PRNKNNPPS-OH (SEQ ID NO: 23); PSKGAPPPS-OH (SEQ ID NO: 24); PSSGAPPPSE-OH (SEQ ID NO: 25); PSSGAPPPS-NH ₂ (SEQ ID NO: 26); PSSGAPPPS- OH (SEQ ID NO: 27); PSSGAPPPSS-OH (SEQ ID NO: 28); PSSGEPPPS-OH (SEQ ID NO: 29); PSSGKKPPS-OH (SEQ ID NO: 30); PSSGKPPPS-NH ₂ (SEQ ID NO : 31); PSSGKPPPS-OH (SEQ ID NO: 32); PSSGPPPS-OH (SEQ ID NO: 33); PSSKAPPPS-OH (SEQ ID NO: 34); PSSKEPPPS-OH (SEQ ID NO: 35); PSSKGAPPPS- OH (SEQ ID NO: 36); PSSKQPPPS-OH (SEQ ID NO: 37); PSSKSPPPS-OH (SEQ ID NO: 38); SGAPPPS-OH (SEQ ID NO: 39); and SSGAPPPS-OH (SEQ ID NO : 40); and (xvii) their combination, provided that the isolated polypeptide with the modified amino acid sequence retains the ability to act as a selective glucagon receptor agonist.

In some embodiments, the isolated polypeptide of the present disclosure includes an amino acid sequence selected from the group consisting of the amino acid sequence of the consensus sequence of SEQ ID NO: 1: X ₁ X ₂ X ₃ GTFTSDX ₁₀ X ₁₁ X _{12 X 13 LX 15 X 16 X 17} X ₁₈ AQEFX ₂₃ X ₂₄ G or T; X ₃ is Q or H; X ₁₀ is Y or _H ; X ₁₁ is S or T; X ₁₂ is K or R; _{X 13} is Y, L or W; It is S, 2-aminoisobutyric acid (Aib), A, E, L, Q, K or I; X ₁₇ is K, E, S, T or A; X ₁₈ is A, R, S, E, L, T or Y _; X ₂₃ is T or V; X ₂₄ is K, I, L or Aib; SEQ ID NO: 4); EPSSGAPPPS-OH (SEQ ID NO: 5); GAPPPS-OH (SEQ ID NO: 6); GGPSSGAPPPS-OH (SEQ ID NO: 7); GPSSGAPPPS-OH (SEQ ID NO: 8) ; KRNKNPPPS-OH (SEQ ID NO: 9); KRNKNPPS-OH (SEQ ID NO: 10); KRNKPPIA-OH (SEQ ID NO: 11); KRNKPPPA-OH (SEQ ID NO: 150); KRNKPPPS-OH (SEQ ID NO: 12); KSSGKPPPS-OH (SEQ ID NO: 13); PESGAPPPS-OH (SEQ ID NO: 14); PKSGAPPPS-OH (SEQ ID NO: 15); PKSKAPPPS-NH ₂ (SEQ ID NO: 16) ; PKSKAPPPS-OH (SEQ ID NO: 17); PKSKEPPPS-NH ₂ (SEQ ID NO: 18); PKSKEPPPS-OH (SEQ ID NO: 19); PKSKQPPPS-OH (SEQ ID NO: 20); PKSKEPPPS-NH ₂ (SEQ ID NO: 21); PSKSKSPPS-OH (SEQ ID NO: 22); PRNKNNPPS-OH (SEQ ID NO: 23); PSKGAPPPS-OH (SEQ ID NO: 24); PSSGAPPPSE-OH (SEQ ID NO: 25 ); PSSGAPPPS-NH ₂ (SEQ ID NO: 26); PSSGAPPPS-OH (SEQ ID NO: 27); PSSGAPPPSS-OH (SEQ ID NO: 28); PSSGEPPPS-OH (SEQ ID NO: 29); PSSGKKPPS-OH (SEQ ID NO: 30); PSSGKPPPS-NH ₂ (SEQ ID NO: 31); PSSGKPPPS-OH (SEQ ID NO: 32); PSSGSPPPS-OH (SEQ ID NO: 33); PSSKAPPPS-OH (SEQ ID NO: 34); PSSKEPPPPS-OH (SEQ ID NO: 35); PSSKGAPPPS-OH (SEQ ID NO: 36); PSSKQPPPS-OH (SEQ ID NO: 37); PSSKSPPPS-OH (SEQ ID NO: 38); SGAPPPS-OH (SEQ ID NO: 39); and SSGAPPPS-OH (SEQ ID NO: 40).

In some embodiments, the isolated polypeptide of the present disclosure includes an amino acid sequence selected from the group consisting of the amino acid sequence of the consensus sequence of SEQ ID NO: 2: X ₁ X ₂ X ₃ GTFTSDX ₁₀ X ₁₁ X _{12 X 13 LX 15 X 16 X 17 _ _} It is Q or H; X ₁₀ is Y or H; X ₁₁ is S or T; _{X 12} is K or R; X ₁₃ is Y, L or W; X ₁₅ is D or E; Butyric acid (Aib), A or S; X ₁₇ is A or K; X ₁₈ is R, S, L or Y; X ₂₄ is K, I or Aib; X ₂₅ is H or W; and the Z tail does not exist or Selected from the group consisting of: PSSGAPPPS- _NH2 (SEQ ID NO:26); PSSGAPPPS-OH (SEQ ID NO:27); and PSKKSPPPS- _NH2 (SEQ ID NO:21).

In some embodiments, the isolated polypeptide of the present disclosure includes an amino acid sequence selected from the group consisting of the amino acid sequence of the consensus sequence of SEQ ID NO: 3: YSX ₃ GTFTSDYSKYLDX ₁₆ X ₁₇ X ₁₈ AQEFVX ₂₄ WLEDE -Z tail-(OH/NH ₂ ) (SEQ ID NO: 3), where: X ₃ is Q or H; X ₁₆ is 2-aminoisobutyric acid (Aib) or A; X ₁₇ is A or K; _{X 18} is R, _S or Y;

In some embodiments, an isolated polypeptide of the present disclosure includes an amino acid sequence selected from the group consisting of: YSHGTFTSDYSKYLD(Aib)KYAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 41), which is described herein Also known as Compound A1; YSHGTFTSDYSKYLD(Aib)KSAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO:42), which is also known herein as Compound A2; YSQGTFTSDYSKYLDAARAQEFVKWLEDEPKSKSPPPS- _NH2 (SEQ ID NO:43), which is herein Also referred to as Compound A3; YSHGTFTSDYSKYLD(Aib)KRAQEFVIWLEDEPSSGAPPPS- _OH (SEQ ID NO: 44), which is also referred to herein as Compound A4; is Compound A5; and WSQGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 46), which is also referred to herein as Compound A6.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLD(Aib)KYAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 41). In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLD(Aib)KSAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 42). In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSQGTFTSDYSKYLDAARAQEFVKWLEDEPKSKSPPPS-NH ₂ (SEQ ID NO: 43). In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLD(Aib)KRAQEFVIWLEDEPSSGAPPPS-OH (SEQ ID NO: 44). In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLDSARAQEFVKWLEDEPSSGAPPPS- _NH2 (SEQ ID NO: 45). In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence WSQGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 46).

In some embodiments, the isolated polypeptide of the present disclosure consists of the amino acid sequence YSHGTFTSDYSKYLD(Aib)KYAQEFV(Aib)WLED EPSSGAPPPS-OH (SEQ ID NO: 41). In some embodiments, the isolated polypeptide of the present disclosure consists of the amino acid sequence YSHGTFTSDYSKYLD(Aib)KSAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 42). In some embodiments, the isolated polypeptide of the present disclosure consists of the amino acid sequence YSQGTFTSDYSKYLDAARAQEFVKWLEDEPKSKSPPPS-NH ₂ (SEQ ID NO: 43). In some embodiments, the isolated polypeptide of the present disclosure consists of the amino acid sequence YSHGTFTSDYSKYLD(Aib)KRAQEFVIWLEDEPSSGAPPPS-OH (SEQ ID NO: 44). In some embodiments, the isolated polypeptide of the present disclosure consists of the amino acid sequence YSHGTFTSDYSKYLDSARAQEFVKWLEDEPSSGAPPPS- _NH2 (SEQ ID NO: 45). In some embodiments, the isolated polypeptide of the present disclosure consists of the amino acid sequence WSQGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 46).

In some embodiments, an isolated polypeptide of the present disclosure includes an amino acid sequence selected from the group consisting of: YSHGTFTSDYSKYLD(Aib)ARAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 47); which is described herein _Also known as Compound A7; NO: 49); which is also referred to herein as Compound A9; YSHGTFTSDYSKYLD (Aib) KRAQEFV (Aib) WLEDEEPSSGAPPPS-OH (SEQ ID NO: 50); which is also referred to herein as Compound A10; YSHGT FTSDYSKYLD (Aib) KRAQEFV (Aib) WLEDEEEPSSGAPPPS-OH (SEQ ID NO: 51); which is also referred to herein as Compound A11; YSHGTFTSDYSKYLD (Aib) KRAQEFV (Aib) WLEDEGAPPPS-OH (SEQ ID NO: 52); which is also referred to herein as Compound A11; Compound A12; ; It is also referred to herein as Compound A14; YSHGTFTSDYSKYLD(Aib)SRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 55); It is also referred to herein as Compound A15; OH (SEQ ID NO: 56); which is also referred to herein as Compound A16; YSHGTFTSDYSKYLD(Aib)ERAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 57); which is also referred to herein as Compound A17; YSHGTFTSDYSKWLD ( Aib)ARAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO:58); which is also referred to herein as Compound A18; YSHGTFTSDYSKWLD(Aib)SRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO:59); which is herein referred to as Also known as Compound A19; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGKPPPS-OH (SEQ ID NO: 60); which is also referred to herein as Compound A20; : 61); It is also referred to as Compound A21 in this article; ) WLEDEPSSKAPPPS-OH (SEQ ID NO: 63); which is also referred to herein as Compound A23; YSHGTFTSDYSKYLD (Aib) KRAQEFV (Aib) WLEDEPSSKGAPPPS-OH (SEQ ID NO: 64); which is also referred to herein as Compound A24 ; YSHGTFTSDYSKYLD (Aib) KRAQEFV (Aib) WLEDEPSSGAPPPSS-OH (SEQ ID NO: 65); which is also referred to herein as compound A25; YSHGTFTSDYSKYLD (Aib) KRAQEFV (Aib) WLEDEPSSGAPPPSE-OH (SEQ ID NO: 66); which Also referred to herein as Compound A26; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEGPSSGAPPPS-OH (SEQ ID NO: 67); which is also referred to herein as Compound A27; SEQ ID NO: 68); which is also referred to herein as Compound A28; YSHGTFTSDYSKYLD (Aib) KRAQEFV (Aib) WLEDEPKSGAPPPS-OH (SEQ ID NO: 69); which is also referred to herein as Compound A29; YSHGTFTSDYSKYLD (Aib) KRAQEFV(Aib)WLEDEPSKGAPPPS-OH (SEQ ID NO:70); which is also referred to herein as Compound A30; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPESGAPPPS-OH (SEQ ID NO:71); which is also referred to herein as Compound A30; is Compound A31; ); which is also referred to herein as Compound A33; YTHGTFTSDHSKWLD(Aib)KRAQ EFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 74); which is also referred to herein as Compound A34; YTHGTFTSDDYSKWLD(Aib)ARAQEFV(Aib) WLEDEPSSGAPPPS-OH (SEQ ID NO: 75); which is also referred to herein as Compound A35; YSHGTFTSDHSKWLD(Aib)ARAQEFV(Aib) WLEDEPSSGAPPPS-OH (SEQ ID NO: 76); which is also referred to herein as Compound A36; YSHGTFTSDYSKYLDSARAQEFVKWLEDEPSSGAPPPS-OH (SEQ ID NO: 77); which is also referred to herein as Compound A37; YSHGTFTSDYSKWLDSARAQEFVKWLEDEPSSGAPPPS-OH (SEQ ID NO: 78); which is also referred to herein as Compound A38; 79); which is also referred to herein as Compound A39; YSHGTFTSDHSKWLDSARAQEFVKWLEDEPSSGAPPPS-OH (SEQ ID NO: 80); which is also referred to herein as Compound A40; YTHGTFTSDHSKWLDEARAQEFVKWLEDEPSSGAPPPS-OH (SEQ ID NO: 81); which is also referred to herein as Compound A40; Referred to as Compound A41; YTHGTFTSDYSKWLDSKRAQEFVKWLEDEPSSGAPPPS-OH (SEQ ID NO: 82); which is also referred to herein as Compound A42; (SEQ ID NO: 84); which is also referred to herein as Compound A44; YSHGTFTSDYSKYLD (Aib) KRAQEFV (Aib) WLEDEKRNKPPPA-OH (SEQ ID NO: 85); which is also referred to herein as Compound A45; YSHGTFTSDYSKYLD (Aib )KRAQEFV(Aib)WLEDEKRNKPPIA-OH(SEQ ID NO:86); which is also referred to herein as Compound A46; Also known as Compound A47; :89); It is also referred to as Compound A49 in this article; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEKRNKPPPS-OH (SEQ ID NO: 90); It is also referred to as Compound A50 in this article; YSHGTFTSDYSKYLDLKRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 91); which is also referred to herein as Compound A51; YSHGTFTSDYSKYLDIKRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 92); which is also referred to herein as Compound A52; YSHGTFTSDYSKYLD(Aib)KRAQEFVLWLEDEPSSGAPPPS-OH (SEQ ID NO:93); which is also referred to herein as Compound A53; )KRAQEFV(Aib)WLEDEPSSKSPPPS-OH (SEQ ID NO:95); which is also referred to herein as Compound A55; Referred to as Compound A56; : 98); It is also referred to as Compound A58 in this article; ) WLEDEPSSGKPPPS-OH (SEQ ID NO: 100); which is also referred to herein as Compound A60; YSHGTFTSDYSKYLDSARAQEFVKWLEDEPSSGKPPPS-OH (SEQ ID NO: 101); which is also referred to herein as Compound A61; ) WLEDEPSSGAPPPS-OH (SEQ ID NO: 102); which is also referred to herein as Compound A62; YTHGTFTSDYSKYLDSARAQEFVKWLEDEPSSGAPPPS-OH (SEQ ID NO: 103); which is also referred to herein as Compound A63; )WLEDEPSSGAPPPS-OH (SEQ ID NO: 104); which is also referred to herein as compound A64; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPKSKEPPPS-NH ₂ (SEQ ID NO: 105); which is also referred to herein as compound _{A65 ;} ); which is _also referred to herein as Compound A67; 109); which is also referred to herein as compound A69; YSHGTFTSDYSKYLDSARAQEFVKWLEDEPKSKSPPPS-OH (SEQ ID NO: 110); which is also referred to herein as compound A70; YSHGTFTSDYSKYLDSARAQEFVKWLEDEPKSKAPPPS-OH (SEQ ID NO: 111); which is also referred to herein as compound A70; Referred to as Compound _A71 ; _SKAPPPS -NH ₂ (SEQ ID NO: 114); which is also referred to herein as Compound A74; WSHGTTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 115); which is also referred to herein as Compound A75; YSHGTFTSDYSKYLD(Aib)KAAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 116); which is also referred to herein as Compound A76; YSHGTFTSDYSKYLD(Aib)KTAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 117); which is Also referred to herein as Compound A77; ID NO: 119); which is also referred to herein as Compound A79; YSHGTFTSDYSKYLDAARAQEFVKWLEDEPKSKSPPPS-NH ₂ (SEQ ID NO: 120); which is also referred to herein as Compound A80; YSQGTFTSDYSKYLDSARAQEFVKWLEDEPKSKSPPPS-OH (SEQ ID NO: 12 1); It is also referred to herein as Compound A81; YSQGTFTSDYSKYLDSARAQEFVKWLEDEPKSKAPPPS-OH (SEQ ID NO: 122); It is also referred to herein as Compound A82; A83; YSHGTFTSDYSKYLDSARAQEFVKHLEDEPKSKSPPPS-OH (SEQ ID NO: 124); which is also referred to _herein as Compound A84; Compound A85; ; which is also referred to herein as Compound A87; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGKKPPS-OH (SEQ ID NO: 128); which is also referred to herein as Compound A88; OH (SEQ ID NO: 129); which is also referred to herein as Compound A89; YSHGTFTSDYSKYLD (Aib) KAAQEFV (Aib) WLEDEPSSGKPPPS-OH (SEQ ID NO: 130); which is also referred to herein as Compound A90; WSQGTFTSDYSKYLD ( Aib) KRAQEFV (Aib) WLEDEPSSGKPPPS-OH (SEQ ID NO: 131); which is also referred to herein as Compound A91; WSQGTFTSDYSKYLD (Aib) KAAQEFV (Aib) WLEDEPSSGAPPPS-OH (SEQ ID NO: 132); which is referred to herein as Compound A91 Also known as Compound A92; WSQGTFTSDYSKYLD(Aib)KAAQEFV(Aib)WLEDEPSSGKPPPS-OH (SEQ ID NO: 133); which is also referred to herein as Compound A93; YSQGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 134); which is also referred to herein as Compound A94; YSQGTFTSDYSKYLD (Aib) KAAQEFV (Aib) WLEDEPSSGAPPPS-OH (SEQ ID NO: 135); which is also referred to herein as Compound A95; and YSQGTFTSDYSKYLD (Aib) KAAQEFV (Aib) WLEDEPSSGKPPPS-OH (SEQ ID NO: 136); which is also referred to herein as Compound A96. In some embodiments, the polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 47 to 136.

In some embodiments, the isolated polypeptide is selected from the group consisting of Compound A1, Compound A2, Compound A3, Compound A4, Compound A5, and Compound A6. In some embodiments, the polypeptide compound A1 is isolated. In some embodiments, the polypeptide compound A2 is isolated. In some embodiments, the polypeptide compound A3 is isolated. In some embodiments, the polypeptide compound A4 is isolated. In some embodiments, the polypeptide compound A5 is isolated. In some embodiments, the polypeptide compound A6 is isolated.

In some embodiments, the isolated polypeptide of the present disclosure includes an amino acid sequence selected from the group consisting of the amino acid sequence of the consensus sequence of SEQ ID NO: 137: YSQGTFTSDYSKYLDSX ₁₇ RAQX ₂₁ FVX ₂₄ WLX ₂₇ X ₂₈ T _-OH (SEQ ID NO _: 137) _, where _: K _* is located in the lactam bridge; X ₂₄ is _K or K** _, where K** is located in the lactam bridge with E or E**, where E** is located in the lactam bridge with K** at X ₂₄ .

In some embodiments, an isolated polypeptide of the present disclosure includes an amino acid sequence selected from the group consisting of: YSQGTFTSDYSKYLDSK*RAQE*FVK**WLDE**T-OH (SEQ ID NO: 138), herein Referred to as Compound A104 and YSQGTFTSDYSKYLDSK*RAQE*FVK**WLQE**T-OH (SEQ ID NO: 139), referred to as Compound A105 herein. In some embodiments, the polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 138 and 139.

Lipophilic substituents are attached to any peptide, optionally via a spacer

In some embodiments, the disclosed peptides are optionally substituted with one or more lipophilic substituents, each optionally via a bivalent spacer.

Conjugation of one or more lipophilic substituents to the disclosure, each optionally via a bivalent spacer, is intended to prolong the action of the peptide by promoting binding to serum albumin and delaying renal clearance of the conjugated peptide. Applicants have discovered that certain disclosed peptides that have affinity for albumin and extend elimination half-life in humans are particularly suitable for use in the disclosed methods of administration via implantable osmotic drug delivery devices.

In some embodiments, the peptides are disclosed to have an elimination half-life (t _1/2 ) of at least about 5 hours following subcutaneous administration to humans. In some embodiments, the peptides are disclosed to have an elimination half-life (t _1/2 ) of at least about 8 hours, 10 hours, 12 hours, 16 hours, 24 hours, or longer following subcutaneous administration to humans.

As used herein, "lipophilic substituent" includes substituents containing 4 to 40 carbon atoms, 8 to 25 carbon atoms, or 12 to 22 carbon atoms. The lipophilic substituent may be attached to the amine group of the peptide (e.g., the epsilon-amine group of a lysine residue) via the carboxyl group of the lipophilic substituent or optionally the amine group of a spacer, which in turn is attached to The amine group of an amino acid (such as lysine) residue forms an amide bond. In some embodiments, the peptide contains three, two, or preferably one lipophilic substituent, each with or without an optional spacer.

In some embodiments, lipophilic substituents comprise linear or branched alkyl groups. In some embodiments, the lipophilic substituent is a acyl group of a linear or branched fatty acid. In some embodiments, the lipophilic substituent is a acyl group of formula CH ₃ (CH ₂ ) _n CO-, wherein n is an integer from 4 to 38, an integer from 4 to 24, such as CH ₃ (CH ₂ ) ₆ CO- , CH ₃ (CH ₂ ) ₈ CO-, CH ₃ (CH ₂ ) ₁₀ CO-, CH ₃ (CH ₂ ) ₁₂ CO-, CH ₃ (CH ₂ ) ₁₄ CO-, CH ₃ (CH ₂ ) ₁₆ CO- , CH ₃ (CH ₂ ) ₁₈ CO-, CH ₃ (CH ₂ ) ₂₀ CO- or CH ₃ (CH ₂ ) ₂₂ CO-. In some embodiments, n is 6, 8, 10, 12, 14, 16, 18, 20, or 22. In some embodiments, the lipophilic substituent is a acyl group of formula CH ₃ (CH ₂ ) ₁₄ CO-.

In some embodiments, the lipophilic substituent is a acyl group of a linear or branched fatty acid, which is further substituted with one or more carboxylic acid and/or hydroxamic acid groups. In some embodiments, the lipophilic substituent is a acyl group of formula HOOC(CH ₂ ) _m CO-, wherein m is an integer from 4 to 38, an integer from 4 to 24, such as HOOC(CH ₂ ) ₁₄ CO-, HOOC (CH ₂ ) ₁₆ CO-, HOOC(CH ₂ ) ₁₈ CO-, HOOC(CH ₂ ) ₂₀ CO- or HOOC(CH ₂ ) ₂₂ CO-. In some embodiments, the lipophilic substituent is HOOC( _CH2 ) _16CO- . In some embodiments, m is 6, 8, 10, 12, 14, 16, 18, 20, or 22.

In some embodiments, the lipophilic substituent is attached to the parent peptide via a bivalent "spacer." In some embodiments, the spacer includes a bivalent group of Formula I: -N(R ₁ )(CHR ₂ ) _p CO-[N(R ₃ )((CH ₂ ) ₂ O(CH ₂ ) ₂ O) _q (CH ₂ )CO-] _r (Formula I)

Wherein each R ₁ and R ₃ are hydrogen or C ₁ -C ₄ alkyl; each R ₂ is H or CO ₂ H; p is 1, 2, 3, 4, 5 or 6; q is 1, 2 or 3; r is 0 or 1, and the spacer forms a bridge between the amine group of the disclosed peptide and the CO-group of the lipophilic substituent.

In some embodiments, each _R1 is hydrogen. In some embodiments, each _R3 is hydrogen. In some embodiments, each R ₁ and each R ₃ is hydrogen.

In some embodiments, at least one _R2 is _CO2H . In some embodiments, one _R2 is _CO2H .

In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3. In some embodiments, p is 4. In some embodiments, p is 5. In some embodiments, p is 6.

In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3.

In some embodiments, r is 0. In some embodiments, r is 1.

In some embodiments, the spacer is gamma-glutamyl, -NH( _CHCO2H )( _CH2 ) _2CO-- . In some embodiments, the spacer is γ-aminobutyryl, i.e. -NH( _CH2 ) _3CO-- . In some embodiments, the spacer is β-aspartamide acyl, i.e., -NH(CHCO ₂ H)(CH ₂ )CO—-. In some embodiments, the spacer is -NH( _CH2 ) _2CO-- . In some embodiments, the spacer is glycinyl. In some embodiments, the spacer is β-propylamine acyl. In some embodiments, a peptide of SEQ ID NO: 151 is provided, wherein the lipophilic substituent is connected to the epsilon-amine group of the lysine acid via a spacer.

In some embodiments, the spacer is -NHCH(CO ₂ H)(CH ₂ ) ₂ CO--[N(R ₃ )((CH ₂ ) ₂ O(CH ₂ ) ₂ O) _q (CH ₂ )CO -] _r . In some embodiments, the spacer is -NH(CH ₂ ) ₃ CO--[N(R ₃ )((CH ₂ ) ₂ O(CH ₂ ) ₂ O) _q (CH ₂ )CO-] _r . In some embodiments, the spacer is -NHCH(CO ₂ H)(CH ₂ ) ₂ CO-NH((CH ₂ ) ₂ O(CH ₂ ) ₂ O) ₂ (CH ₂ )CO-. In some embodiments, the spacer is -NH( _CH2 ) _3CO -NH(( _CH2 ) _2O ( _CH2 ) _2O ) ₂ ( _CH2 )CO-. In some embodiments, the spacer is -NHCH(CO ₂ H)CH ₂ CO--[N(R ₃ )((CH ₂ ) ₂ O(CH ₂ ) ₂ O) _q (CH ₂ )CO-] _r . In some embodiments, the spacer is -NH(CH ₂ ) ₂ CO--[N(R ₃ )((CH ₂ ) ₂ O(CH ₂ ) ₂ O) _q (CH ₂ )CO-] _r .

In some embodiments, the spacer is an amino acid (eg, Lys, Glu, or Asp) or a dipeptide such as Gly-Lys. In some embodiments, when the spacer is Lys, Glu or Asp, one carboxyl group of the spacer can form a amide bond with the amine group of the disclosed peptide, and the amine group of the spacer can form a amide bond with the carboxyl group of the lipophilic substituent. Amine bond.

In some embodiments, lipophilic substituents are combined with spacers to form the structure of Formula II:

In some embodiments, lipophilic substituents are combined with spacers to form the structure of Formula III:

In some embodiments, each lipophilic substituent is attached, optionally via a spacer, to the epsilon-amine group of a lysine residue contained in the parent peptide.

In some embodiments, the isolated polypeptide of the present disclosure includes an amino acid sequence selected from the group consisting of the amino acid sequence of the consensus sequence of SEQ ID NO: 151: X ₁ SX ₃ GTFTSDX ₁₀ SKYLDX ₁₆ X ₁₇ X ₁₈ AQX ₂₁ FVX ₂₄ WLEDEPX ₃₁ SX ₃₃ X ₃₄ PPPS-OH (SEQ ID NO: 151), where: X1=Y or W; X3=H or Q; , S, Aib, K or K***; X17=A, K, Aib or K***; X18=Y, S or R; X21=E, K or K***; K or K***; X31=S, K or K***; X33=G, K or K***; and The ε-amine group of the acid side chain is optionally covalently linked via a spacer to a lipophilic substituent, both as defined herein.

In some embodiments, K*** is a lysine acid, and the ε-amine group of the lysine acid side chain is covalently linked to a lipophilic substituent via a spacer. In some embodiments, K*** is a lysine acid, and the ε-amine group of the lysine acid side chain is covalently linked to a lipophilic substituent in the absence of a spacer (ie, no spacer is required).

In some embodiments, the isolated polypeptide of the present disclosure includes an amino acid sequence selected from the group consisting of the amino acid sequence of the consensus sequence of SEQ ID NO: 151, wherein: X1 = Y or W; X3 = H or Q; X10=Y or K***; X16=A, S, Aib or K***; X17=A, K or Aib; X24=I, Aib or K***; X31=S or K***; X33=G, K or K***; and X34=A or S; where K*** is lysine, and the ε-amine of lysine acid side chain The group is optionally covalently linked via a spacer to a lipophilic substituent, both as defined herein.

In some embodiments, K*** is a lysine acid, and the ε-amine group of the lysine acid side chain is covalently linked to a lipophilic substituent via a spacer. In some embodiments, K*** is a lysine acid, and the ε-amine group of the lysine acid side chain is covalently linked to a lipophilic substituent in the absence of a spacer (ie, no spacer is required).

In some embodiments, X1=Y. In some embodiments, X1=W.

In some embodiments, X3=H. In some embodiments, X3=Q.

In some embodiments, X10=Y. In some embodiments, X10=K. In some embodiments, X10=K***.

In some embodiments, X16=A. In some embodiments, X16=S. In some embodiments, X16=Aib. In some embodiments, X16=K. In some embodiments, X16=K***.

In some embodiments, X17=A. In some embodiments, X17=K. In some embodiments, X17=Aib. In some embodiments, X17=K***.

In some embodiments, X18=Y. In some embodiments, X18=S. In some embodiments, X18=R.

In some embodiments, X21=E. In some embodiments, X21=K. In some embodiments, X21=K***.

In some embodiments, X24=1. In some embodiments In, X24=Aib. In some embodiments, X24=K. In some embodiments, X24=K***.

In some embodiments, X31=S. In some embodiments, X31=K. In some embodiments, X31=K***.

In some embodiments, X33=G. In some embodiments, X33=K. In some embodiments, X33=K***.

In some embodiments, X16 is K***; in some embodiments, X21 is K***; in some embodiments, X24 is K***; in some embodiments, X33 is K** *, where K*** is lysine, and the ε-amine group of lysine is covalently connected to the lipophilic substituent and spacer of formula II:

In some embodiments, X16 is K***; in some embodiments, X21 is K***; in some embodiments, X24 is K***; in some embodiments, X33 is K** *, where K*** is lysine, and the ε-amine group of lysine is covalently connected to the lipophilic substituent and spacer of formula III:

In some embodiments, one or more of X10, X16, X17, X21, X24, X31, or Covalently linked lipophilic substituents and spacers of formula II.

In some embodiments, one or more of X10, X16, X17, X21, X24, X31, or Covalently linked lipophilic substituents and spacers of formula III.

In some embodiments, one of X10, X16, X17, X21, X24, X31, or Lipophilic substituents and spacers of connecting formula II.

In some embodiments, one of X10, X16, X17, X21, X24, X31, or Lipophilic substituent and spacer connecting formula III.

In some embodiments, pharmaceutical compositions are provided comprising any of the disclosed peptides formulated as a trifluoroacetate, acetate, or hydrochloride salt. In some embodiments, pharmaceutical compositions are provided comprising any of the disclosed peptides formulated as a trifluoroacetate salt. In some embodiments, pharmaceutical compositions are provided comprising any of the disclosed peptides formulated as an acetate salt. In some embodiments, pharmaceutical compositions are provided comprising any of the disclosed peptides formulated as a hydrochloride salt.

In some embodiments, there is provided a pharmaceutical composition comprising any of the disclosures formulated as a trifluoroacetate, acetate or hydrochloride salt, and the peptide comprises an amino acid sequence selected from the consensus sequence of SEQ ID NO: 151 A group of amino acid sequences. In some embodiments, there are provided pharmaceutical compositions comprising any of the disclosed peptides formulated as a trifluoroacetate salt, and the peptide comprises an amine group selected from the group consisting of the amino acid sequence of the consensus sequence of SEQ ID NO: 151 acid sequence. In some embodiments, there are provided pharmaceutical compositions comprising any of the disclosed peptides formulated as an acetate salt, and the peptide comprises an amino acid sequence selected from the group consisting of the amino acid sequence of the consensus sequence of SEQ ID NO: 151 . In some embodiments, there are provided pharmaceutical compositions comprising any of the disclosed peptides formulated as a hydrochloride salt, and the peptide comprises an amino acid selected from the group consisting of the amino acid sequence of the consensus sequence of SEQ ID NO: 151 sequence. In some embodiments, in the peptide of SEQ ID NO: 151, one of X10, X16, X17, X21, X24, X31, or X33 is K***, wherein K*** is lysine, and The ε-amine group of lysine acid is covalently connected to the lipophilic substituent and spacer of formula II. In some embodiments, in the peptide of SEQ ID NO: 151, one of X10, X16, X17, X21, X24, X31, or X33 is K***, wherein K*** is lysine, and ε- of lysine The amine group is covalently linked to the lipophilic substituent and spacer of formula III.

In some embodiments, any isolated polypeptide of the present disclosure is provided, wherein the polypeptide comprises one or more amino acid residues, each of the one or more amino acid residues optionally covalently bound via a spacer A lipophilic substituent is attached, both as defined herein. In some embodiments, each of the one or more amino acid residues is covalently linked to a lipophilic substituent and spacer of Formula II or III.

In some embodiments, any of the isolated polypeptides of the present disclosure, wherein the polypeptide comprises at least one lysine residue, wherein the epsilon-amine group of the lysine side chain is optionally covalently linked to a lipophilic substitution via a spacer Base, both as defined herein. In some embodiments, at least one lysine residue each has an epsilon-amine group covalently linked to the lipophilic substituent and spacer of Formula II or III.

In some embodiments, the isolated polypeptide is selected from the group consisting of compounds B1 to B48.

In some embodiments, the isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLD(Aib)KYAQEFV(Aib)WLEDEPSSK****APPPS-OH (SEQ ID NO: 152), which is also referred to herein as Compound B1 .

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLD(Aib)KYAQEFVK****WLEDEPSSGAPPPS-OH (SEQ ID NO: 153), which is also referred to herein as Compound B2.

In some embodiments, the isolated polypeptides of the present disclosure comprise an amine The amino acid sequence YSHGTFTSDYSKYLDK****KYAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 154), which is also referred to herein as compound B3.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDK****SKYLD(Aib)KYAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 155), which is also referred to herein as Compound B4 .

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLD(Aib)KYAQEFV(Aib)WLEDEPSSK***** APPPS-OH (SEQ ID NO: 156), which is also referred to herein as a compound B5.

In some embodiments, the isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLD(Aib)KYAQEFVK*****WLEDEPSSGAPPPS-OH (SEQ ID NO: 157), which is also referred to herein as Compound B6.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLDK*****KYAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 158), which is also referred to herein as Compound B7.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDK*****SKYLD(Aib)KYAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 159), which is also referred to herein as a compound B8.

In some embodiments, the isolated polypeptides of the present disclosure comprise an amine The amino acid sequence YSHGTFTSDYSKYLD(Aib)KSAQEFV(Aib)WLEDEPSSK****APPPS-OH (SEQ ID NO: 160), which is also referred to herein as compound B9.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLD(Aib)KSAQEFVK****WLEDEPSSGAPPPS-OH (SEQ ID NO: 161), which is also referred to herein as Compound B10.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLDK****KSAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 162), which is also referred to herein as Compound B11.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDK****SKYLD(Aib)KSAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 163), which is also referred to herein as Compound B12 .

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLD(Aib)KSAQEFV(Aib)WLEDEPSSK***** APPPS-OH (SEQ ID NO: 164), which is also referred to herein as a compound B13.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLD(Aib)KSAQEFVK*****WLEDEPSSGAPPPS-OH (SEQ ID NO: 165), which is also referred to herein as Compound B14.

In some embodiments, the isolated polypeptides of the present disclosure comprise an amine The amino acid sequence YSHGTFTSDYSKYLDK*****KSAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 166), which is also referred to herein as compound B15.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDK*****SKYLD(Aib)KSAQEFV(Aib)WLEDEPSSGAPPP S-OH (SEQ ID NO: 167), which is also referred to herein as Compound B16.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence WSQGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSK****APPPS-OH (SEQ ID NO: 168), which is also referred to herein as Compound B17 .

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence WSQGTFTSDYSKYLD(Aib)KRAQEFVK****WLEDEPSSGAPPPS-OH (SEQ ID NO: 169), which is also referred to herein as Compound B18.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence WSQGTFTSDYSKYLDK****KRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 170), which is also referred to herein as Compound B19.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence WSQGTFTSDK****SKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 171), which is also referred to herein as Compound B20 .

In some embodiments, the isolated polypeptides of the present disclosure comprise an amine The amino acid sequence WSQGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSK***** APPPS-OH (SEQ ID NO: 172), which is also referred to herein as compound B21.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence WSQGTFTSDYSKYLD(Aib)KRAQEFVK*****WLEDEPSSGAPPPS-OH (SEQ ID NO: 173), which is also referred to herein as Compound B22.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence WSQGTFTSDYSKYLDK*****KRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 174), which is also referred to herein as Compound B23.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence WSQGTFTSDK*****SKYLD(Aib)KRAQEFV(Aib)WLEDEPSS GAPPPS-OH (SEQ ID NO: 175), which is also referred to herein as Compound B24.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLD(Aib)KRAQEFVIWLEDEPSSK****APPPS-OH (SEQ ID NO: 176), which is also referred to herein as Compound B25.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLDK****KRAQEFVIWLEDEPSSGAPPPS-OH (SEQ ID NO: 177), which is also referred to herein as Compound B26.

In some embodiments, the isolated polypeptides of the present disclosure comprise an amine The amino acid sequence YSHGTFTSDK****SKYLD(Aib)KRAQEFVIWLEDEPSSGAPPPS-OH (SEQ ID NO: 178), which is also referred to herein as compound B27.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLD(Aib)KRAQK****FVIWLEDEPSSGAPPPS-OH (SEQ ID NO: 179), which is also referred to herein as Compound B28.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLD(Aib)KRAQEFVIWLEDEPSSK*****APPPS-OH (SEQ ID NO: 180), which is also referred to herein as Compound B29.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLD(Aib)KRAQK*****FVIWLEDEPSSGAPPPS-OH (SEQ ID NO: 181), which is also referred to herein as Compound B30.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLDK*****KRAQEFVIWLEDEPSSGAPPPS-OH (SEQ ID NO: 182), which is also referred to herein as Compound B31.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDK*****SKYLD(Aib)KRAQEFVIWLEDEPSSGAPPPS-OH (SEQ ID NO: 183), which is also referred to herein as Compound B32.

In some embodiments, an isolated polymorph of the present disclosure includes the amino acid sequence YSQGTFTSDYSKYLDAARAQEFVKWLEDEPKSK****SPPPS-NH _{2 (} SEQ ID NO: 184), which is also referred to herein as compound B33.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSQGTFTSDYSKYLDAARAQEFVK****WLEDEPKSKSPPPS-NH _{2 (} SEQ ID NO: 185), which is also referred to herein as Compound B34.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSQGTFTSDYSKYLDK****ARAQEFVKWLEDEPKSKSPPPS-NH _{2 (} SEQ ID NO: 186), which is also referred to herein as Compound B35.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSQGTFTSDK****SKYLDAARAQEFVKWLEDEPKSKSPPPS-NH _{2 (} SEQ ID NO: 187), which is also referred to herein as Compound B36.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSQGTFTSDYSKYLDAARAQEFVKWLEDEPKSK*****SPPPS-NH _{2 (} SEQ ID NO: 188), which is also referred to herein as Compound B37.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSQGTFTSDYSKYLDAARAQEFVK*****WLEDEPKSKSPPPS-NH _{2 (} SEQ ID NO: 189), which is also referred to herein as Compound B38.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSQGTFTSDYSKYLDK*****ARAQEFVKWLEDEPKSKSPPPS-NH _{2 (} SEQ ID NO: 190), which is also referred to herein as compound B39.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSQGTFTSDK*****SKYLDAARAQEFVKWLEDEPKSKSPPPS-NH _{2 (} SEQ ID NO: 191), which is also referred to herein as Compound B40.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSQGTFTSDK*****SKYLDAARAQEFVKWLEDEPKSKSPPPS-NH _{2 (} SEQ ID NO: 191), which is also referred to herein as Compound B41.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLDSARAQEFVK****WLEDEPSSGAPPPS-NH _{2 (} SEQ ID NO: 193), which is also referred to herein as Compound B42.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLDK****ARAQEFVKWLEDEPSSGAPPPS-NH _{2 (} SEQ ID NO: 194), which is also referred to herein as Compound B43.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDK****SKYLDSARAQEFVKWLEDEPSSGAPPPS-NH _{2 (} SEQ ID NO: 195), which is also referred to herein as Compound B44.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLDSARAQEFVKWLEDEPSSK*****APPPS-NH _{2 (} SEQ ID NO: 196), which is also referred to herein as Compound B45.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLDSARAQEFVK*****WLEDEPSSGAPPPS-NH _{2 (} SEQ ID NO: 197), which is also referred to herein as Compound B46.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDYSKYLDK*****ARAQEFVKWLEDEPSSGAPPPS-NH _{2 (} SEQ ID NO: 198), which is also referred to herein as Compound B47.

In some embodiments, an isolated polypeptide of the present disclosure includes the amino acid sequence YSHGTFTSDK*****SKYLDSARAQEFVKWLEDEPSSGAPPPS-NH _{2 (} SEQ ID NO: 199), which is also referred to herein as Compound B48.

In the above embodiment, each ε-amine group of the lysine residue of the indicated peptide residue (K****) is covalently linked to the carbonyl group of Formula II to form an amide:

In the above embodiment, each ε-amine group of the lysine residue of the indicated peptide residue (K*****) is covalently linked to the carbonyl group of Formula III to form an amide:

In some embodiments, the isolated polypeptides of the present disclosure comprise an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 152 to SEQ ID NO: 199.

In some embodiments, the isolated polypeptide of the present disclosure is selected from the group consisting of Compound B1 to Compound B8.

In some embodiments, the isolated polypeptide of the present disclosure is selected from the group consisting of Compound B9 to Compound B16.

In some embodiments, the isolated polypeptide of the present disclosure is selected from the group consisting of Compound B17 to Compound B24.

In some embodiments, the isolated polypeptide of the present disclosure is selected from the group consisting of Compound B25 to Compound B32.

In some embodiments, the isolated polypeptide of the present disclosure is selected from the group consisting of Compound B33 to Compound B40.

In some embodiments, the isolated polypeptide of the present disclosure is selected from the group consisting of Compound B41 to Compound B48.

In some embodiments, the isolated polypeptides of the present disclosure comprise SEQ ID NO: 154, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162 , SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 185, SEQ ID NO: 188, SEQ ID NO: 189, SEQ The amino acid sequence of the group consisting of the amino acid sequences of ID NO: 190 and SEQ ID NO: 192.

In some embodiments, an isolated polypeptide of the present disclosure includes an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 160, SEQ ID NO: 165, and SEQ ID NO: 166.

In some embodiments, the isolated polypeptide of the present disclosure is selected from the group consisting of Compound B1 to Compound B48.

In some embodiments, the isolated polypeptide of the present disclosure is selected from the group consisting of Compound B3, Compound B6, Compound B7, Compound B9, Compound B10, Compound B11, Compound B14, Compound B15, Compound B17, Compound B21, Compound B22, Compound B34 , the group consisting of compound B37, compound B38, compound B39 and compound B41.

In some embodiments, the isolated polypeptide of the present disclosure is selected from the group consisting of Compound B9, Compound B14 and Compound B15.

In some embodiments, the isolated polypeptide of the present disclosure is Compound B9. In some embodiments, the isolated polypeptide of the present disclosure is Compound B14. In some embodiments, the isolated polypeptide of the present disclosure is Compound B15.

In some embodiments, the isolated polypeptide of the present disclosure includes an amino acid sequence selected from the group consisting of the amino acid sequences of Compound B1 to Compound B48.

2.0.2 Peptide combinations

In some embodiments, the isolated glucagon analog polypeptide of the present disclosure, which is a selective glucagon receptor agonist, is combined with a compound selected from the group consisting of acidotropin, exenatide, exenatide derivatives, A second dose combination of the group consisting of exenatide analogues, glucagon-like peptide-1 (GLP-1), GLP-1 derivatives and GLP-1 analogues was co-formulated. In some embodiments, any glucagon analog of the present disclosure is co-modulated in combination with exenatide.

In some embodiments, any glucagon analog of the present disclosure is formulated in combination with exenatide, wherein the glucagon analog is not covalently linked to lipophilic substituents and spacers as described herein.

In some embodiments, the glucagon analog of the present disclosure is formulated in combination with exenatide, wherein the glucagon analog is selected from the group consisting of compounds A1 to A105. In some embodiments, the glucagon analog of the present disclosure is formulated in combination with exenatide, wherein the glucagon analog is selected from the group consisting of compounds A1, A2, A3, A4, A5 and A6. In some embodiments, the glucagon analog of the present disclosure is formulated in combination with exenatide, wherein the glucagon analog is Compound A1. In some embodiments, the glucagon analog of the present disclosure is formulated in combination with exenatide, wherein the glucagon analog is Compound A2. In some embodiments, the glucagon analog of the present disclosure is formulated in combination with exenatide, wherein the glucagon analog is Compound A3. In some embodiments, the glucagon analog of the present disclosure is formulated in combination with exenatide, wherein the glucagon analog is Compound A4. In some embodiments, the glucagon analog of the present disclosure is formulated in combination with exenatide, wherein the glucagon analog is Compound A5. In some embodiments, the glucagon analog of the present disclosure is formulated in combination with exenatide, wherein the glucagon analog is Compound A6.

In some embodiments, any glucagon analog of the present disclosure is formulated in combination with exenatide, wherein the glucagon analog has at least one amine covalently linked to a lipophilic substituent and spacer as described herein Basic acid. In some embodiments, any glucagon analog of the present disclosure is formulated in combination with exenatide, wherein the glucagon analog has at least one lipophilic substituent and spacer covalently linked to Formula II or III. The lysine residue of the ε-amine group.

In some embodiments, the glucagon analog of the present disclosure is formulated in combination with exenatide, wherein the glucagon analog is selected from the group consisting of compounds B1 to B48. In some embodiments, any glucagon analog of the present disclosure is formulated in combination with exenatide, wherein the glucagon analog is selected from the amino acid sequence of SEQ ID NO: 152 to SEQ ID NO: 199 The group formed.

In some embodiments, any glucagon analog of the present disclosure is combined with exenatide in a fixed ratio of glucagon analog: exenatide of 1000:1 to 1:1000. In some embodiments, any glucagon analog of the present disclosure is combined with exenatide in a fixed ratio of glucagon analog: exenatide of 500:1 to 1:500. In some implementations In this example, any glucagon analog of the present disclosure is prepared in combination with exenatide at a fixed ratio of glucagon analog to exenatide of 100:1 to 1:100. In some embodiments, any glucagon analog of the present disclosure is combined with exenatide in a fixed ratio of glucagon analog to exenatide of 50:1 to 1:50. In some embodiments, any glucagon analog of the present disclosure is combined with exenatide in a fixed ratio of glucagon analog: exenatide of 25:1 to 1:25. In some embodiments, any glucagon analog of the present disclosure is combined with exenatide in a fixed ratio of glucagon analog to exenatide of 10:1 to 1:10. In some embodiments, any glucagon analog of the present disclosure is combined with exenatide in a fixed ratio of glucagon analog to exenatide of 1:1 to 1:10. In some embodiments, any glucagon analog of the present disclosure is combined with exenatide in a fixed ratio of glucagon analog to exenatide of 10:1 to 1:1. In some embodiments, any glucagon analog of the present disclosure is combined with exenatide in a fixed ratio of glucagon analog to exenatide of 5:1 to 1:5. In some embodiments, any glucagon analog of the present disclosure is combined with exenatide in a fixed ratio of glucagon analog to exenatide of 1:1 to 1:5. In some embodiments, any glucagon analog of the present disclosure is combined with exenatide in a fixed ratio of glucagon analog: exenatide from 5:1 to 1:1.

The present invention also provides methods of treating type 2 diabetes in an individual in need of treatment. The method includes providing one or more isolated glucagon receptor selective agonist polypeptides of the present disclosure. In some embodiments, the method comprises providing a self-osmotic delivery device for continuous delivery of an isolated glucagon receptor-selective agonist. An agonist polypeptide, wherein substantially steady-state delivery of a therapeutic concentration of an isolated glucagon-specific agonist polypeptide is achieved within a period of about 7 days or less after implantation of the osmotic delivery device in the individual. The self-osmotic delivery device delivers substantially steady-state delivery of an isolated glucagon-specific agonist polypeptide over a continuous period of administration. Human beings are the preferred subjects for implementing the present invention.

In some embodiments of the invention, the administration period is, for example, at least about 3 months, at least about 3 months to about one year, at least about 4 months to about one year, at least about 5 months to about one year, at least About 6 months to about one year, at least about 8 months to about one year, at least about 9 months to about one year, at least about 10 months to about one year, at least about one year to about two years, at least about Two years to about three years.

In some embodiments of the invention, substantially steady-state delivery of a therapeutic concentration of an isolated glucagon-specific agonist polypeptide is performed within about 5 days or less after implantation of the osmotic delivery device in the individual. About 4 days or less after the osmotic delivery device is implanted in the individual, about 3 days or less after the individual is implanted with the osmotic delivery device, about 2 days or less after the individual is implanted with the osmotic delivery device, or in the individual Achieved in approximately 1 day or less after implantation of the osmotic delivery device. In preferred embodiments of the present invention, substantially steady-state delivery of therapeutic concentrations of an isolated glucagon-specific agonist polypeptide occurs within about 2 days or less, and more preferably, after implantation of the osmotic delivery device in the individual. It is achieved in about 1 day or less.

In a further embodiment, the treatment methods of the present invention provide a significant reduction in the subject's fasting plasma glucose concentration (relative to the subject's fasting plasma glucose concentration prior to the subject's implantation of the osmotic delivery device) after the subject has implanted the osmotic delivery device. Approximately 7 days or after insertion into the osmotic delivery device Within a shorter period of time, about 6 days or less after the individual is implanted with the osmotic delivery device, about 5 days or less after the individual is implanted with the osmotic delivery device, about 4 days after the individual is implanted with the osmotic delivery device or less, about 3 days or less after the individual is implanted with the osmotic delivery device, about 2 days or less after the individual is implanted with the osmotic delivery device, or about 3 days or less after the individual is implanted with the osmotic delivery device Achieved in 1 day or less. In preferred embodiments of the present invention, a significant reduction in an individual's fasting plasma glucose concentration after implantation of the osmotic delivery device relative to the individual's fasting plasma glucose concentration prior to implantation is achieved in about 2 days or less, preferably in about 2 days or less. Achieved within about 1 day or less after implantation of the osmotic delivery device in the individual, or more preferably within about 1 day after implantation of the osmotic delivery device in the individual. A significant decrease in fasting plasma glucose generally means that it is statistically significant through appropriate statistical test analysis or is determined to be significant for an individual by a licensed physician. Significant reductions in fasting plasma glucose relative to preimplantation baseline were generally maintained during the dosing period.

In yet further embodiments of the first aspect of the invention, the method of treatment further comprises terminating the ability to continuously deliver the glucagon-specific agonist polypeptide such that the glucagon-specific agonist polypeptide is present in the individual's blood sample. At a concentration that is about 6 glucagon-specific agonist polypeptide half-lives or less after cessation of continuous delivery, about 5 glucagon-specific agonist polypeptide half-lives or less after cessation of continuous delivery time, about 4 glucagon-specific agonist polypeptide half-lives or less after terminating continuous delivery, or about 3 glucagon-specific agonist polypeptide half-lives or less after terminating continuous delivery The system is essentially undetectable over time. Glucagon-specific agonist polypeptides can be determined by, for example, RIA, chromatography, ECL assay, ELISA or IEMA detection. Termination of continuous delivery can be accomplished, for example, by removing the osmotic delivery device from the subject.

In a related embodiment of the invention, the method of treatment further comprises the ability to terminate continuous delivery of the glucagon receptor selective agonist polypeptide such that the concentration of the polypeptide in the individual's blood sample after discontinuation of continuous delivery is less than about 72 Within hours, less than about 48 hours after terminating continuous delivery, less than about 24 hours after terminating continuous delivery, less than about 18 hours after terminating continuous delivery, less than about 14 hours after terminating continuous delivery , is substantially undetectable for less than about 12 hours after terminating continuous delivery, less than about 6 hours after terminating continuous delivery, or less than about 4 hours after terminating continuous delivery. In preferred embodiments, glucagon is produced in less than about 24 hours after terminating continuous delivery, within less than about 18 hours after terminating continuous delivery, or preferably within less than about 14 hours after terminating continuous delivery. The specific agonist polypeptide is substantially undetectable in the individual's blood sample.

In some embodiments, the glucagon receptor selective agonist polypeptide is formulated as a suspension formulation. In some embodiments, the suspension formulation includes a particulate formulation comprising a glucagon receptor selective agonist polypeptide and a vehicle formulation. In some embodiments, the glucagon receptor selective agonist polypeptide comprises an isolated polypeptide of the present disclosure, a peptide analog thereof, or a peptide derivative thereof. In some embodiments, the glucagon receptor selective agonist polypeptide comprises an amino acid sequence encompassed by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 137 Amino acid sequence. In some embodiments, the glucagon receptor selective agonist polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4 to 139.

In all aspects of the present invention regarding methods of treating type 2 diabetes, suspension formulations for use in the methods may include, for example, particulate formulations containing an isolated glucagon receptor selective agonist polypeptide and a vehicle. agent formula.

The reservoir of the osmotic delivery device may comprise, for example, titanium or titanium alloys.

In a fifth aspect, the invention relates to methods of treating a disease or condition in an individual in need of treatment. The method includes providing continuous delivery of a drug from an osmotic delivery device, wherein substantially steady-state delivery of a therapeutic concentration of the drug is achieved within a period of about 7 days or less after implantation of the osmotic delivery device in the individual. The self-osmotic delivery device delivers substantially steady-state drug delivery for a continuous administration period of at least about 3 months. A drug has a known or determined half-life in the average individual. Human beings are the preferred subjects for implementing the present invention. The present invention includes drugs effective in treating a disease or condition as well as osmotic delivery devices containing drugs in the present methods for treating a disease or condition in an individual in need thereof. Advantages of the present invention include attenuating spike-related drug toxicity and attenuating sub-optimal drug therapy associated with low concentrations.

In some embodiments of the invention, the administration period is, for example, at least about 3 months, at least about 3 months to about one year, at least about 4 months to about one year, at least about 5 months to about one year, at least About 6 months to about one year, at least about 8 months to about one year, at least about 9 months to about one year, or at least about 10 months to about one year.

In some embodiments of this aspect of the invention, substantially steady-state delivery of the therapeutic concentration of the drug is performed approximately 100 seconds after implantation of the osmotic delivery device in the individual. Within a period of 7 days or less, Within a period of about 5 days or less after the individual is implanted with an osmotic delivery device, Within a period of about 4 days or less after the individual is implanted with an osmotic delivery device, Within a period of about 4 days or less after the individual is implanted with an osmotic delivery device Achieved in about 3 days or less, in about 2 days or less after the subject is implanted with the osmotic delivery device, or in about 1 day or less after the subject is implanted with the osmotic delivery device.

In some embodiments of this aspect of the invention, after implantation of the osmotic delivery device in an individual, establishing substantially steady-state delivery of therapeutic concentrations of the drug may require an extended period of time, such as about 2 weeks or less. period, or less than approximately 6 half-lives of the drug in the individual after implantation of the device.

The present invention also provides a method of promoting weight loss in an individual in need thereof, a method of treating overweight or obesity in an individual in need thereof, and/or a method of suppressing appetite in an individual in need thereof. The method includes providing delivery of an isolated glucagon receptor selective agonist polypeptide. In some embodiments, the isolated glucagon receptor selective agonist polypeptide is delivered continuously from an osmotic delivery device, wherein substantially steady-state delivery of the therapeutic concentration of the glucagon receptor selective agonist polypeptide is in the individual This is achieved within a time period of approximately 7 days or less after implantation of the osmotic delivery device. The self-osmotic delivery device delivers substantially steady-state delivery of the glucagon receptor-selective agonist polypeptide over a continuous period of administration. Human beings are the preferred subjects for implementing the present invention. The present invention includes isolated glucagon receptor selective agonist polypeptides and osmotic delivery devices comprising isolated glucagon receptor selective agonist polypeptides for use in the present methods in an individual in need of treatment. The individual may have type 2 diabetes. Individuals in need may have a baseline HbA1c% greater than 10.0%, ie, high baseline (HBL) individuals. Individual first You may not have received medications used to treat type 2 diabetes before.

In some embodiments of the invention, the administration period is, for example, at least about 3 months, at least about 3 months to about one year, at least about 4 months to about one year, at least about 5 months to about one year, at least About 6 months to about one year, at least about 8 months to about one year, at least about 9 months to about one year, or at least about 10 months to about one year, at least about one year to about two years, or At least about two years to about three years.

In a further embodiment, the treatment methods of the present invention provide a significant reduction in the subject's fasting plasma glucose concentration (relative to the subject's fasting plasma glucose concentration prior to the subject's implantation of the osmotic delivery device) after the subject has implanted the osmotic delivery device. About 7 days or less after implantation of the osmotic delivery device, About 6 days or less after implantation of the osmotic delivery device in the individual, About 5 days or less after implantation of the osmotic delivery device in the individual, In the individual About 4 days or less after implantation of the osmotic delivery device, about 3 days or less after implantation of the osmotic delivery device in the individual, about 2 days or less after implantation of the osmotic delivery device in the individual, or Achieved in approximately 1 day or less after implantation of the osmotic delivery device in the individual. In preferred embodiments of the present invention, a significant reduction in an individual's fasting plasma glucose concentration after implantation of the osmotic delivery device relative to the individual's fasting plasma glucose concentration prior to implantation is achieved in about 2 days or less, preferably in about 2 days or less. Achieved within about 1 day or less after implantation of the osmotic delivery device in the individual, or more preferably within about 1 day after implantation of the osmotic delivery device in the individual. A significant decrease in fasting plasma glucose generally means that it is statistically significant through appropriate statistical test analysis or is determined to be significant for an individual by a licensed physician. Significance of fasting plasma glucose relative to pre-implantation baseline The reduction is generally maintained during the investment period.

In all aspects of the present invention regarding methods of treating a disease or condition in an individual, exemplary osmotic delivery devices include the following: an impermeable reservoir including interior and exterior surfaces and first and second open ends; A semipermeable membrane in a sealing relationship with the first open end of the storage tank; a permeation engine located in the storage tank and adjacent to the semipermeable membrane; and a piston adjacent to the permeation engine, wherein the piston is movable with the interior surface of the storage tank a seal, the piston divides the storage tank into a first chamber and a second chamber, the first chamber containing the osmosis engine; a drug formula or a suspension formula containing the drug, wherein the second chamber contains the drug formula or suspension formula and the The drug formula or suspension formula is flowable; and a diffusion adjuster inserted into the second open end of the storage tank, the diffusion adjuster adjacent to the suspension formula. In preferred embodiments, the reservoir contains titanium or a titanium alloy.

In all aspect embodiments of the present invention regarding methods of treating a disease or condition in an individual, the pharmaceutical formulation may include a drug and vehicle formulation. Alternatively, suspension formulations may be used in the methods and may include, for example, particulate formulations containing the drug and vehicle formulations. Vehicle formulations used to form the suspension formulations of the present invention may include, for example, solvents and polymers.

The reservoir of the osmotic delivery device may comprise, for example, titanium or titanium alloys.

In embodiments of all aspects of the invention, implanted osmotic delivery devices may be used to provide subcutaneous delivery.

In embodiments of all aspects of the invention, continuous delivery may be, for example, zero-order, controlled continuous delivery.

Any of the above aspects and embodiments may be combined with any other aspects or embodiments disclosed herein.

3.0.0 Formula and composition

Drugs used in the practice of the present invention are generally uniformly suspended, dissolved or dispersed in a suspension vehicle to form a suspension formulation.

The isolated polypeptides of the present disclosure (also referred to herein as "active compounds") and their derivatives, fragments, analogs and homologs can be incorporated into pharmaceutical compositions suitable for administration. Such compositions generally include a fusion protein and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein is intended to include any and all solvents, dispersion media, coating agents, antibacterial and antifungal agents that are compatible with pharmaceutical administration. , isotonic agents, absorption delaying agents, and the like. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, which is the standard reference text in the field and is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer’s solution, glucose solution and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutical active substances is well known in the art. Unless any conventional media or agents are incompatible with the active compound, their use in the compositions is contemplated. Supplementary active compounds may also be incorporated into the compositions.

The pharmaceutical compositions of the present invention are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral such as intravenous, Intradermal, subdermal, subcutaneous, oral (e.g., inhaled), transdermal (i.e., topical), transmucosal, transrectal, or a combination thereof. In some embodiments, the pharmaceutical composition is formulated for administration via a device or other suitable delivery mechanism suitable for subdermal or subcutaneous implantation and subcutaneous delivery of the pharmaceutical composition. In some embodiments, the pharmaceutical composition is formulated for administration via an implantable device suitable for subdermal or subcutaneous implantation and for subcutaneous delivery of the pharmaceutical composition. In some embodiments, the pharmaceutical composition is formulated for administration via an osmotic delivery device suitable for subdermal or subcutaneous placement or other implantation and subcutaneous delivery of the pharmaceutical composition, such as an implantable osmotic delivery device. Solutions or suspensions for parenteral application, intradermal application, subdermal application, subcutaneous application or combinations thereof may include the following components: sterile diluent such as water for injection, saline solution, fixed oil, polyethylene glycol , glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate , citrate or phosphate, and agents used to adjust osmotic properties such as sodium chloride or dextrose. The pH can be adjusted using acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be enclosed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (which are water-soluble) or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, sterile water, Cremophor EL ^™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be in a fluid form that can be easily poured. The composition must remain stable under the conditions of manufacture and storage and must be preserved in a manner that prevents the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium, including, for example, water, ethanol, polyols (eg, glycerin, propylene glycol, liquid polyethylene glycol, and the like), and appropriate mixtures thereof. Proper fluidity can be maintained, for example, by using coating agents such as lecithin, by maintaining the desired particle size in the dispersion, and by using surfactants. Protection against microorganisms can be achieved by using various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases it is preferred that the composition includes an isotonic agent such as sugar, polyol (such as mannitol), sorbitol or sodium chloride. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that prolongs absorption, such as aluminum monostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating the required amount of the active compound, and optionally one or more of the above ingredients, in an appropriate solvent, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. Taking sterile powders for the preparation of sterile injectable solutions as an example, preparation methods include vacuum drying and freeze drying to produce powders of the active ingredient and any additional desired ingredients from their previously filtered sterilized solutions.

Oral compositions usually include an inert diluent or edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound may be combined with excipients and used in the form of tablets, troches, or capsules. Oral compositions may also be administered The carrier is prepared for use as a mouthwash, wherein the compound in the fluid carrier is applied to the mouth and used to rinse the mouth, and then spit or swallowed. Pharmaceutically compatible binding agents and/or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges and the like may contain any of the following ingredients or compounds of similar nature: binders such as microcrystalline cellulose, tragacanth or gelatin; excipients such as starch or lactose; disintegrating agents such as Alginic acid, Primogel or cornstarch; lubricant such as magnesium stearate or Sterotes; glidant such as colloidal silica; sweetener such as sucrose or saccharin; or flavoring agent such as mint, methyl salicylate or orange flavoring .

For administration by inhalation, the compound is delivered as a gaseous spray from a pressurized container or dispenser containing a suitable propellant (eg, a gas such as carbon dioxide) or a nebulizer.

Systemic administration can also be by transmucosal or transdermal devices. For transmucosal or transdermal administration, penetrating agents suitable for penetrating the barrier are used in the formulation. Such penetrating agents are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels or creams as are generally known in the art.

The compounds may also be prepared in the form of suppositories (eg, using conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compound may be combined with the compound to prevent They are prepared with carriers that are rapidly cleared from the body, such as controlled release formulations, including implants and microencapsulated release systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations will be readily apparent to those of ordinary skill in the art. Materials are also available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions can also be used as pharmaceutically acceptable carriers. These may be prepared according to methods known to those of ordinary skill in the art, such as those described in US Pat. No. 4,522,811.

It is particularly advantageous to formulate oral or parenteral compositions into dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the individuals intended to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired treatment in association with the required pharmaceutical carrier. Effect. The specifications for dosage unit forms of the present invention will be determined and directly dependent upon the unique characteristics of the active compound and the particular therapeutic effect sought to be achieved, combined with the limitations inherent in the art of administering the active compound to the treatment of individuals.

The pharmaceutical composition may be included in a container, package, or dispenser along with instructions for administration.

3.1.0 Drug granule formulation

In one aspect, the present invention provides pharmaceutical particle formulations for pharmaceutical use. Granular formulations typically contain the drug and include one or more stabilizing components (also referred to herein as "excipients"). stable group Examples include, but are not limited to, carbohydrates, antioxidants, amino acids, buffers, inorganic compounds, and surfactants.

In any embodiment, the particulate formulation can include about 50 wt% to about 90 wt% drug, about 50 wt% to about 85 wt% drug, about 55 wt% to about 90 wt% drug, about 60 wt% to about 90 wt% drug, about 65 wt% to about 90 wt% drug. About 85 wt% drug, about 65 wt% to about 90 wt% drug, about 70 wt% to about 90 wt% drug, about 70 wt% to about 85 wt% drug, about 70 wt% to about 80 wt% drug, or about 70 wt% to about 75 wt% drug .

In any embodiment, the granular formulation includes a drug as described above and one or more stabilizers. Stabilizers may be, for example, carbohydrates, antioxidants, amino acids, buffers, inorganic compounds, or surfactants. The amount of stabilizer in a granular formulation can be determined experimentally based on the activity of the stabilizer and the desired characteristics of the formulation in light of the teachings of this specification.

Generally speaking, the amount of carbohydrate in a formula is determined by aggregation considerations. Generally, the amount of carbohydrate should not be too high to avoid promoting crystal growth in the presence of water due to excess non-drug-bound carbohydrate.

Generally speaking, the amount of antioxidants in the formulation is determined by oxidation considerations, whereas the amount of amino acids in the formulation is determined by oxidation considerations and/or particle formability during spray drying.

Generally speaking, the amount of buffer in the formulation is determined by pre-treatment considerations, stability considerations, and particle formability during spray drying. When all stabilizers are dissolved, buffers may be needed to stabilize the drug during processing such as solution preparation and spray drying.

Examples of carbohydrates that may be included in granular formulations include, but are not limited to, monosaccharides (such as fructose, maltose, galactose, glucose, D-mannose, and sorbose), disaccharides (such as lactose, sucrose, trehalose, and fiber disaccharides). sugar), polysaccharides (such as raffinose, miltose, maltodextrin, dextran and starch) and alditol (acyclic polyol; such as mannitol, xylitol, hydrogenated maltose, lactitol, Xylitol, sorbitol, peranosylsorbitol and myo-inositol). Suitable carbohydrates include disaccharides and/or non-reducing sugars such as sucrose, trehalose and raffinose.

Examples of antioxidants that may be included in granular formulations include, but are not limited to, methionine, ascorbic acid, sodium thiosulfate, catalase, platinum, ethylenediaminetetraacetic acid (EDTA), citric acid, cysteine, Thioglycerin, thioglycolic acid, thiosorbitol, butylated hydroxyanisole, butylated hydroxycresol and propyl gallate. In addition, easily oxidized amino acids can be used as antioxidants, such as cysteine, methionine and tryptophan.

Examples of amino acids that may be included in granular formulations include, but are not limited to, arginine, methionine, glycine, histidine, alanine, L-leucine, glutamic acid, isoleucine , L-threonine, 2-aniline, valine, norvaline, proline, phenylalanine, tryptophan, serine, aspartic acid, cysteine, tyrosine, Lysine and norleucine. Suitable amino acids include those that are susceptible to oxidation, such as cysteine, methionine and tryptophan.

Examples of buffers that may be included in granular formulations include, but are not limited to, citrate, histidine, succinate, phosphate, maleate, tris(hydroxymethyl)aminomethane (TRIS), Acetate, carbohydrate Materials and glycine-glycine. Suitable buffers include citrate, histidine, succinate and TRIS.

Examples of inorganic compounds that may be included in the particle formulation include, but _are not limited to, NaCl, _Na2SO4 , _NaHCO3 , _KCl , _KH2PO4 , CaCl2 _, and _MgCl2 .

Additionally, granular formulations may include other stabilizers/excipients such as surfactants and salts. Examples of surfactants include, but are not limited to, Polysorbate 20, Polysorbate 80, ^PLURONIC® (BASF Corporation, Mount Olive, NJ) F68, and sodium dodecyl sulfate (SDS). Examples of salts include, but are not limited to, sodium chloride, calcium chloride, and magnesium chloride.

3.1.1 Exemplary drugs

Pharmaceutical granule formulations contain pharmaceuticals. A drug may be any physiologically or pharmaceutically active substance, particularly one known to be deliverable to the human or animal body.

Suitable drugs include, but are not limited to, peptides, proteins, polypeptides or synthetic analogs of these species and mixtures thereof.

In one embodiment, preferred drugs include macromolecules. Such macromolecules include, but are not limited to, pharmaceutically active peptides, proteins or polypeptides. A number of peptides, proteins or polypeptides useful in practicing the invention are described herein. In addition to the peptides, proteins or polypeptides described, modifications of these peptides, proteins or polypeptides are known to those of ordinary skill in the art and can be used to practice the invention in accordance with the guidance presented herein. Such modifications include, but do not Limited to amino acid analogs, amino acid mimetics, analog polypeptides or derivative polypeptides. Additionally, the drugs disclosed herein may be formulated or administered individually or in combination (eg, using drug mixtures or multiple devices; US Patent Publication No. 2009/0202608).

Some embodiments of the invention include the use of the polypeptides of SEQ ID NOs: 4 to 136, 138, 139, and 143 to 149.

Some embodiments of the invention include the use of a glucagon receptor selective agonist polypeptide in combination with a second polypeptide, such as the following non-limiting examples: insulinotropic peptides, peptide hormones such as glucagon and incretin mimetics (such as GLP-1 and exenatide) and their peptide analogs and peptide derivatives; PYY (also known as peptide YY, peptide tyrosine tyrosine) and its peptides Analogs and peptide derivatives, such as PYY (3-36); acidotropin and its peptide analogs and peptide derivatives); and gastrophin (GIP) and its peptide analogs and peptide derivatives.

GLP-1 (including the three peptide forms GLP-1(1-37), GLP-1(7-37) and GLP-1(7-36)amide) and peptide analogs of GLP-1 have been shown to stimulate insulin secretion (i.e., is insulinotropic), thus inducing cellular glucose uptake and resulting in a decrease in serum glucose concentration (see, eg, Mojsov, S., Int. J. Peptide Protein Research, 40: 333-343 (1992)).

Many GLP-1 peptide derivatives and peptide analogs exhibiting insulinotropic effects are known in the art (see, for example, U.S. Patent Nos. 5,118,666; 5,120,712; 5,512,549; 5,545,618; 5,574,008; 5,574,008; 5,614,492; 5,958,909; 6,191,102; 6,26 8,343; 6,329,336; 6,451,974; 6,458,924; 6,514,500;6,593,295;6,703,359;6,706,689;6,720,407;6,821,949;6,849,708;6,849,714;6,887,470;6,887,849;6,903,186;7,022,674;7 ,041,646; 7,084,243; 7,101,843; 7,138,486; 7,141,547; 7,144,863; and 7,199,217) and clinical trials (such as taspoglutide ) and albiglutide). An example of a GLP-1 peptide derivative useful in practicing the present invention is Victoza® (Novo Nordisk A/S, Bagsvaerd DK) (liraglutide; U.S. Patent Nos. 6,268,343, 6,458,924 and 7,235,627). Once-daily injectable Victoza® (liraglutide) is commercially available in the United States, Europe, and Japan. For ease of reference herein, the family of GLP-1 peptides, GLP-1 peptide derivatives and GLP-1 peptide analogs with insulinotropic activity is collectively referred to as "GLP-1".

The molecule exenatide has the amino acid sequence of secretin-4 (Kolterman O.G., et al., J. Clin. Endocrinol. Metab. 88(7): 3082-9 (2003)) and is chemically synthesized or reorganize performance production. Twice-daily injections of exenatide are commercially available in the United States and Europe and are sold under the brand name Byetta® (Amylin Pharmaceuticals, Inc., San Diego, Calif.). Secretin-3 and secretin-4 are known in the art and were originally isolated from Heloderma spp. (Eng, J., et al., J. Biol. Chem., 265: 20259-62 (1990); Eng., J., et al., J. Biol. Chem., 267: 7402-05 (1992)). Insectin-3 and Insectin-4 are used to treat type 2 diabetes and prevent hyperglycemia. Uses have been proposed (see, eg, US Pat. No. 5,424,286). Many exenatide peptide derivatives and peptide analogs (including, for example, secretin-4 agonists) are within the art (see, for example, U.S. Patent Nos. 5,424,286; 6,268,343; 6,329,336; 6,506,724; 6,514,500; 6,528,486; 6,593,295; 6,703,359; 6,706,689;6,767,887;6,821,949;6,849,714;6,858,576;6,872,700;6,887,470;6,887,849;6,924,264;6,956,026;6,989,366;7,022,674;7 ,041,646; 7,115,569; 7,138,375; 7,141,547; 7,153,825; and 7,157,555) known. An example of an exenatide derivative useful in practicing the present invention is lixisenatide (also known as ZP10, AVE0010) (see, eg, US Pat. No. 6,528,486), which is in clinical trials. For convenience of reference herein, exenatide peptides (including, for example, secretagogue-3, secretagogue-4, and secretagogue-4-amide), exenatide derivatives, and exenatide The family of peptide analogs is collectively called "exenatide".

Peptide YY (PYY) is a peptide amide with 36 amino acid residues. PYY inhibits intestinal motility and blood flow (Laburthe, M., Trends Endocrinol Metab. 1(3): 168-74 (1990), mediates intestinal secretion (Cox, H.M., et al., Br J Pharmacol 101 (2): 247-52(1990); Playford, R.J., et al., Lancet 335(8705):1555-7(1990)) and stimulate net absorption (MacFayden, R.J., et al., Neuropeptides 7(3):219-27 (1986)). Two major in vivo variants, PYY(1-36) and PYY(3-36), have been identified (e.g. Eberlein, G. A., et al., Peptides 10(4),797-803(1989)). The sequences of PYY and its peptide analogs and peptide derivatives are known in the art (eg, US Pat. Nos. 5,574,010 and 5,552,520).

Acretin is a naturally occurring 37-amino acid peptide hormone found in the colon that has been found to suppress appetite and promote weight loss (Wynne K, et al., Int J Obes (Lond) 30(12): 1729-36( 2006)). The sequences of modulin and its peptide analogs and peptide derivatives are known in the art (for example, Bataille D, et al., Peptides 2 (Suppl 2): 41-44 (1981); and U.S. Patent Publication No. 2005/ 0070469 and 2006/0094652).

Gastric inhibitory peptide (GIP) is an insulin-stimulating peptide hormone (Efendic, S., et al., Horm Metab Res. 36: 742-6 (2004)) and is produced by the duodenal and jejunal mucosa in response to absorbed fat and stimulates the pancreas Secreted by carbohydrates that secrete insulin. GIP circulates in the body as a biologically active 42-amino acid peptide. GIP is also known as glucose-dependent insulinotropic protein. GIP is a 42-amino acid gastrointestinal regulatory peptide that stimulates pancreatic beta cells to secrete insulin in the presence of glucose (Tseng, C., et al., PNAS 90: 1992-1996 (1993)). The sequences of GIP and its peptide analogs and peptide derivatives are known in the art (for example, Meier J.J., Diabetes Metab Res Rev. 21(2):91-117 (2005) and Efendic S., Horm Metab Res. 36 (11-12):742-6(2004)).

Glucagon is a peptide hormone produced by pancreatic alpha cells, which increases blood glucose concentration. Its effect is opposite to that of insulin, which lowers glucose concentrations. When glucose levels in the blood fall too low, the pancreas releases glucagon. Lift Glycosin causes the liver to convert stored glycogen into glucose and release it into the bloodstream. High blood glucose concentrations stimulate insulin release. Insulin allows glucose to be absorbed and used by insulin-dependent tissues. Therefore, glucagon and insulin are part of the feedback system that keeps blood sugar concentrations at a stable level.

Glucagon-like peptide-2 (GLP-2) is a 33 amino acid peptide, and the human sequence is HADGSFSDEMNTILDNLAARDFINWLIQTKITD (SEQ ID NO: 200). GLP-2 is produced by a specific post-translational proteolytic cleavage of proglucagon, which also releases the related glucagon-like peptide-1 (GLP-1). GLP-2 is produced by endocrine L cells in the small intestine and various neurons in the central nervous system. Small intestinal GLP-2 is secreted together with GLP-1 during nutrient intake. When administered externally, GLP-2 produces multiple effects in humans and rodents, including small intestinal growth, increased small intestinal function, reduced bone breakdown, and neuroprotective effects. GLP-2 acts in an endocrine manner to link intestinal growth and metabolism with nutrient intake.

Examples of half-lives for some peptides are as follows: exenatide approximately 2.5 hours; GLP-1 approximately 2 minutes; GIP approximately 5 minutes; PYY approximately 8 minutes; glucagon approximately 6 minutes; acidotropin approximately 6 minutes; and GLP-2 approximately 6 minutes.

Drugs may also take a variety of forms, including, but not limited to, the following: uncharged molecules; components of molecular complexes; and pharmaceutically acceptable salts such as hydrochlorides, hydrobromides, sulfates, laurates, palmates, etc. acid salt, phosphate, nitrate, borate, acetate, maleate, tartrate, oleate or salicate. In the case of acidic drugs, salts of metal, amine or organic cations such as quaternary ammonium may be used. In addition, simple derivatives of the drug having solubility characteristics suitable for the purposes of the present invention such as esters, Ethers, amides, and the like may also be used herein.

The above-mentioned drugs and other drugs known to those of ordinary skill in the art can be used in methods of treating a variety of conditions, including but not limited to the following: chronic pain, hemophilia and other blood diseases, endocrinological diseases, metabolic diseases , non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), Alzheimer's disease, cardiovascular diseases (such as heart failure, atherosclerosis, and acute tubular syndrome), rheumatic diseases, Diabetes (including type 1, type 2, human immunodeficiency virus therapy-induced, adult latent autoimmune diabetes, and steroid-induced), asymptomatic hypoglycemia, restrictive lung disease, chronic obstructive pulmonary disease, fat Atrophy, metabolic syndrome, leukemia, hepatitis, renal failure, infectious diseases (including bacterial infections, viral infections (such as human immunodeficiency virus, hepatitis C virus, hepatitis B virus, yellow fever virus, West Nile virus, dengue virus, Marburg virus, and Ebola virus), and parasitic infections), genetic diseases (such as cerebral glycoside deficiency and adenosine deaminase deficiency), hypertension, septic shock, autoimmune diseases (e.g., Graves disease (Grave's disease, systemic lupus erythematosus, multiple sclerosis and rheumatoid arthritis), shock and wasting disease, cystic fibrosis, lactose intolerance, Crohn's disease, inflammatory bowel disease, gastrointestinal cancer ( Including colon and rectal cancer), breast cancer, leukemia, lung cancer, bladder cancer, kidney cancer, non-Hodgkin's lymphoma, pancreatic cancer, thyroid cancer, endometrial cancer and other cancers. Additionally, some of the above agents may be used to treat infectious diseases requiring chronic treatment, including but not limited to tuberculosis, malaria, leishmaniasis, trypanosomiasis (sleeping sickness and Chagas' disease), and parasites.

The amount of drug in the drug particle formulation is that amount required to deliver a therapeutically effective amount of the agent to achieve the desired therapeutic outcome in the individual to whom the drug is delivered. In practice, this will vary depending on such variables as the specific agent, severity of the condition, and desired therapeutic effect. Beneficial agents and their dosage unit amounts are known in the art; see Goodman & Gilman's The Pharmacological Basis of Therapeutics, 11th Ed., (2005), McGraw Hill; Remington's Pharmaceutical Sciences, 18th Ed., (1995), Mack Publishing Co.; and Martin's Physical Pharmacy and Pharmaceutical Sciences, 1.00 edition (2005), Lippincott Williams & Wilkins. In addition, highly concentrated drug particles are described in US Patent Publication No. 2010/0092566. Generally speaking, for an osmotic delivery system, the volume of the chamber containing the drug formulation is between about 100 μl and about 1000 μl, and more preferably between about 140 μl and about 200 μl. In one embodiment, the chamber volume containing the drug formulation is approximately 150 μl.

The pharmaceutical particle formulation of the present invention is chemically and physically stable at the delivery temperature for preferably at least 1 month, preferably at least 3 months, more preferably at least 6 months, more preferably at least 12 months. The delivery temperature is generally normal human body temperature, such as about 37°C, or slightly higher, such as about 40°C. In addition, the pharmaceutical granule formulation of the present invention is chemically and physically stable at storage temperature for preferably at least 3 months, preferably at least 6 months, and more preferably at least 12 months. Examples of storage temperatures include refrigeration temperature, such as about 5°C; or room temperature, such as about 25°C.

The drug particle formulation may be considered chemically stable if at the delivery temperature after about 3 months, preferably after about 6 months, preferably after about 12 months, and After about 6 months, after about 12 months, and preferably after about 24 months, less than about 25% is formed at storage temperature; preferably less than about 20%, more preferably less than about 15%, more preferably Less than about 10% and more preferably less than about 5% of the drug particles are decomposition products.

A drug particle formulation may be considered physically stable if it forms less than about 10% after about 3 months, preferably about 6 months at delivery temperature and about 6 months, preferably about 12 months at storage temperature. , preferably less than about 5%, more preferably less than about 3%, more preferably less than 1% of the aggregate of the drug.

When the drug in the drug particle formulation is protein, the protein solution is stored in a frozen state and freeze-dried or spray-dried into a solid state. Tg (glass transition temperature) can be a factor that should be considered when achieving stable protein compositions. While not intending to be bound by any particular theory, the theory of forming high Tg amorphous solids to stabilize peptides, polypeptides or proteins has been used in the pharmaceutical industry. Typically, if an amorphous solid has a higher Tg, such as 100°C, the peptide product will not be mobile when stored at room temperature or even 40°C because the storage temperature is below the Tg. Calculations using Molecular Information have shown that if the glass transition temperature is above the storage temperature of 50°C, the molecule has zero mobility. Zero mobility of a molecule is associated with better stability. Tg also depends on the moisture concentration in the product formulation. Generally, the more moisture, the lower the Tg of the composition.

Therefore, in some aspects of the invention, excipients with higher Tg can be included in protein formulations to improve stability, such as sucrose (Tg=75°C.) and trehalose (Tg=110°C). Preferably, the granular formulation may be prepared using methods such as spray drying, freeze drying, drying, freeze drying, grinding, granulation, ultrasonic drop creation, condensation. The particles are formed from a mixture of components by crystallization, precipitation or other technical processes available in the art to form particles. In one embodiment of the invention, the particles are spray dried. Particles preferably have substantially uniform shape and size.

Particles are generally of a size such that they can be delivered via implantable osmotic delivery devices. Uniform particle shape and size generally helps provide a consistent and uniform rate of release from the delivery device; however, particle formulations with non-normal particle size distribution characteristics may also be used. For example, in a typical implantable osmotic delivery device having a delivery hole, the particle size is less than about 30%, more preferably less than about 20%, more preferably less than about 10% of the diameter of the delivery hole. In one embodiment of the particulate formulation for use with an osmotic delivery system, in which the delivery pore diameter of the implant is about 0.5 mm, the particle size may be, for example, less than about 150 microns to about 50 microns. In one embodiment of the particulate formulation for use with an osmotic delivery system, in which the delivery pore diameter of the implant is about 0.1 mm, the particle size may be, for example, less than about 30 microns to about 10 microns. In one embodiment, the pores are about 0.25 mm (250 microns) and the particle size is about 2 microns to about 5 microns.

One of ordinary skill in the art will understand that particle populations follow the principles of particle size distribution. Widely used, art-recognized methods of describing particle size distribution include, for example, mean diameter and D values, such as the _D50 value, which is often used to represent the mean diameter of the particle size range for a given sample.

The particles of the particle formulation have a diameter of between about 2 microns and about 150 microns, such as less than 150 microns in diameter, less than 100 microns in diameter. diameter, less than 50 microns in diameter, less than 30 microns in diameter, less than 10 microns in diameter, less than 5 microns in diameter, and about 2 microns in diameter. Preferably, the particles have a diameter of between about 2 microns and about 50 microns.

The particles of the particle formulation comprising the isolated glucagon-specific agonist polypeptide have an average diameter of between about 0.3 microns and about 150 microns. The particles of the particle formulation comprising an isolated glucagon-specific agonist polypeptide have an average diameter of between about 2 microns and about 150 microns, such as an average diameter of less than 150 microns, an average diameter of less than 100 microns, an average diameter of less than 50 microns. Mean diameter, mean diameter less than 30 microns, mean diameter less than 10 microns, mean diameter less than 5 microns, and mean diameter about 2 microns. In some embodiments, the particles have an average diameter of between about 0.3 microns and 50 microns, such as between about 2 microns and about 50 microns. In some embodiments, the particles have an average diameter of between 0.3 microns and 50 microns, such as between about 2 microns and about 50 microns, with the diameter of each particle being less than about 50 microns.

Generally speaking, when incorporated into a suspending vehicle, the particles of the granular formulation do not settle in less than about 3 months, preferably do not settle in less than about 6 months, and more preferably do not settle in less than about 12 months at the delivery temperature. Settles within a month, preferably does not settle in less than about 24 months, and optimally does not settle at delivery temperatures in less than about 36 months. Suspension vehicles generally have a viscosity of between about 5,000 and about 30,000 poise, preferably between about 8,000 and about 25,000 poise, and more preferably between about 10,000 and about 20,000 poise. In one embodiment, the suspension vehicle has a viscosity of about 15,000 poise (plus or minus about 3,000 poise). Generally speaking, smaller particles tend to have lower settling rates in viscous suspension vehicles than larger particles. Therefore, micron to nanometer sized particles Generally speaking, the grain can do whatever you want. In viscous suspension formulations, based on simulation model testing, particles of the present invention ranging from about 2 microns to about 7 microns will not settle at room temperature for at least 20 years. In one embodiment of the particle formulation for an implantable osmotic delivery device of the present invention, particles having a size in the range of less than about 50 microns, more preferably less than about 10 microns, and more preferably from about 2 microns to about 7 microns are included. Particles.

In one embodiment, the drug particle formulation includes the drug as described above, one or more stabilizers and optionally a buffer. Stabilizers may be, for example, carbohydrates, antioxidants, amino acids, buffers, inorganic compounds, or surfactants. The amounts of stabilizers and buffers in granular formulations can be determined experimentally based on the activity of the stabilizers and buffers and the desired characteristics of the formulation in light of the teachings of this specification. Generally speaking, the amount of carbohydrate in a formula is determined by aggregation considerations. Generally, the amount of carbohydrate should not be too high to avoid promoting crystal growth in the presence of water due to excess non-drug-bound carbohydrate. Generally speaking, the amount of antioxidants in the formulation is determined by oxidation considerations, whereas the amount of amino acids in the formulation is determined by oxidation considerations and/or particle formability during spray drying. Generally speaking, the amount of buffer in the formulation is determined by pre-treatment considerations, stability considerations, and particle formability during spray drying. Buffers may be needed to stabilize the drug during processing such as solution preparation and spray drying when all excipients are dissolved.

Examples of carbohydrates that may be included in granular formulations include, but are not limited to, monosaccharides (such as fructose, maltose, galactose, glucose, D-mannose, and sorbose), disaccharides (such as lactose, sucrose, trehalose, and fiber disaccharides). sugars), polysaccharides (e.g. raffinose, meltriose, maltodextrin, dextran and starch) and alditols (acyclic polyols; e.g. mannitol, xylose alcohol, hydrogenated maltose, lactitol, xylitol, sorbitol, peranosylsorbitol and inositol). Preferred carbohydrates include disaccharides and/or non-reducing sugars such as sucrose, trehalose and raffinose.

Examples of antioxidants that may be included in granular formulations include, but are not limited to, methionine, ascorbic acid, sodium thiosulfate, catalase, platinum, ethylenediaminetetraacetic acid (EDTA), citric acid, cysteine, Thioglycerin, thioglycolic acid, thiosorbitol, butylated hydroxyanisole, butylated hydroxycresol and propyl gallate. In addition, easily oxidized amino acids can be used as antioxidants, such as cysteine, methionine and tryptophan.

Examples of amino acids that may be included in granular formulations include, but are not limited to, arginine, methionine, glycine, histidine, alanine, L-leucine, glutamic acid, isoleucine , L-threonine, 2-aniline, valine, norvaline, proline, phenylalanine, tryptophan, serine, aspartic acid, cysteine, tyrosine, Lysine and norleucine.

Examples of buffering agents that may be included in granular formulations include, but are not limited to, citrates, histidines, succinates, phosphates, maleates, TRIS, acetates, carbohydrates, and glycine-glycinate. amino acids.

Examples of inorganic compounds that may be included in the particle formulation include, but are not limited to, NaCl, _Na2SO4 , _{NaHCO3 , KCl} , _KH2PO4 , CaCl2 _, and _MgCl2 .

In addition, granule formulations may include other excipients such as surfactants and salts. Examples of surfactants include, but are not limited to, Polysorbate 20, Polysorbate 80, PLURONIC® (BASF Corporation, Mount Olive, N.J.) F68, and Sodium Lauryl Sulfate (SDS). Examples of salts include, but are not limited to, sodium chloride, calcium chloride, and magnesium chloride.

All ingredients included in the granular formulation are acceptable for medical use in mammals in general and in humans in particular.

Briefly, the selected drug or drug combination is formulated into a solid dry powder that retains maximum chemical and biological stability of the drug. The particulate formulation provides long-term storage stability at elevated temperatures, thus allowing for the delivery of stable and bioavailable drugs to individuals over extended periods of time.

3.2.0 Vehicle formula and suspension formula

In one aspect, the suspending vehicle provides a stable environment in which the drug particulate formulation is dispersed. The drug particle formulation is chemically and physically stable in the suspension vehicle (as described above). Suspension vehicles generally include one or more polymers and one or more solvents that form a solution with sufficient viscosity to uniformly suspend the drug-containing particles. The suspending vehicle may further comprise components including, but not limited to, surfactants, antioxidants, and/or other compounds that are soluble in the vehicle.

The viscosity of the suspending vehicle is generally sufficient to prevent settling of the drug particulate formulation during storage and use in delivery methods (eg, in implantable osmotic delivery devices). The suspending vehicle is biodegradable, in which the suspending vehicle disintegrates or decomposes in response to the biological environment within a period of time, while the drug particles are dissolved in the biological environment and the active pharmaceutical ingredient (i.e., the drug) in the particles is absorbed.

In embodiments, the suspending medium is a "single-phase" suspending medium. Agent, which is a solid, semi-solid or liquid homogeneous system that is physically and chemically uniform as a whole.

The solvent used to dissolve the polymer can affect the characteristics of the suspension formulation, such as the behavior of the drug particle formulation during storage. The solvent may be selected in combination with the polymer so that the resulting suspension vehicle exhibits phase separation when exposed to an aqueous environment. In some embodiments of the present invention, the solvent and polymer combination may be selected such that the resulting suspension vehicle exhibits phase separation when exposed to an aqueous environment having less than about 10% water.

The solvent may be an acceptable solvent that is immiscible with water. The solvent may also be selected such that the polymer is soluble in the solvent at high concentrations, such as a polymer concentration of greater than about 30%. Examples of solvents useful in practicing the present invention include, but are not limited to, lauryl alcohol, benzyl benzoate, benzyl alcohol, lauryl lactate, decanol (also known as decyl alcohol), ethylhexyl lactate Esters, and long chain (C ₈ to C ₂₄ ) aliphatic alcohols, esters, or mixtures thereof. The solvent used in the suspending vehicle may be "dry", that is, it has a low water content. Preferred solvents for use in suspension vehicle formulations include lauryl lactate, lauryl alcohol, benzyl benzoate, and mixtures thereof.

Examples of polymers used to prepare the suspension vehicle of the present invention include, but are not limited to, polyesters (such as polylactic acid and polylactic acid polyglycolic acid), polymers containing pyrrolidone (such as polyethylene with a molecular weight of about 2,000 to about 1,000,000 pyrrolidone), esters or ethers of unsaturated alcohols (such as vinyl acetate), polyoxyethylene polyoxypropylene block copolymers, or mixtures thereof. Polyvinylpyrrolidone can be characterized by its viscosity index K value (for example, K-17). In one embodiment, the polymer system has a strength of 2,000 to 1,000,000 Subweight polyvinylpyrrolidone. In a preferred embodiment, the polymer is polyvinylpyrrolidone K-17 (generally having an approximate average molecular weight of 7,900 to 10,800). The polymers used in the suspending vehicle may include one or more different polymers or may include different grades of a single polymer. The polymers used in the suspending vehicle can also be anhydrous or have a low water content.

Generally speaking, the composition of the suspending vehicle used in the present invention can vary based on the desired performance characteristics. In one embodiment, the suspension vehicle may include about 40 wt% to about 80 wt% polymer and about 20 wt% to about 60 wt% solvent. Preferred embodiments of the suspension vehicle include a vehicle formed from a combination of polymer and solvent in the following ratios: about 25 wt% solvent and about 75 wt% polymer; about 50 wt% solvent and about 50 wt% polymer; about 75wt% solvent and about 25wt% polymer. Thus, in some embodiments, the suspending vehicle may comprise and, in other embodiments, consist essentially of selected components.

Suspension media can exhibit Newtonian behavior. The suspending vehicle is generally formulated to provide a viscosity that maintains uniform dispersion of the particulate formulation over a predetermined period of time. This helps facilitate customization of suspension formulations to provide controlled delivery of drugs contained in drug particle formulations. The viscosity of the suspending vehicle can vary depending on the intended application, the size and type of particle formulation, and the content of the particle formulation in the suspending vehicle. The viscosity of the suspending vehicle can be varied by changing the type or relative amounts of solvents or polymers used.

The suspension vehicle may have a viscosity ranging from about 100 poise to about 1,000,000 poise, preferably from about 1,000 poise to about 100,000 poise. In preferred embodiments, the suspension vehicle generally has a viscosity of from about 5,000 to about 30,000 poise, preferably from about 8,000 to about 25,000 poise, and more preferably from about 10,000 to about 20,000 poise at 33°C. . In one embodiment, the suspension vehicle has a viscosity of about 15,000 poise (plus or minus about 3,000 poise) at 33°C. Viscosity can be measured using a parallel plate rheometer at 33°C and a shear rate of 10 ^-4 /sec.

Suspension vehicles can exhibit phase separation when exposed to an aqueous environment; however, in general, suspension vehicles do not exhibit substantial phase separation as a function of temperature. For example, suspension vehicles generally do not exhibit phase separation at temperatures in the range of about 0°C to about 70°C and upon temperature cycling, such as cycling from 4°C to 37°C to 4°C.

Suspension vehicles can be prepared by combining polymer and solvent under anhydrous conditions, such as in a dry box. The polymer and solvent can be combined at elevated temperatures, such as from about 40°C to about 70°C, and allowed to liquefy and form a single phase. The ingredients can be blended under vacuum to remove air bubbles from the anhydrous ingredients. The ingredients may be combined using a conventional mixer (such as a double helix blade or similar mixer) set at a speed of approximately 40 rpm. However, higher speeds can also be used to mix the ingredients. Once a liquid solution of the ingredients is achieved, the suspending vehicle can be cooled to room temperature. Differential scanning calorimetry (DSC) can be used to verify that the suspension vehicle is single-phase. Additionally, vehicle components (eg, solvents and/or polymers) can be treated to substantially reduce or substantially remove peroxide (eg, by methionine treatment; see, eg, U.S. Patent Application Publication 2007-0027105 ).

The drug particulate formulation is added to the suspending vehicle to form a suspended formulation. In some embodiments, the suspension formulation may comprise a drug particle formulation and a suspending vehicle and in other embodiments consist essentially of a drug particle formulation and a suspending vehicle. Composition of media.

Suspension formulations can be prepared by dispersing the granular formulation in a suspending vehicle. The suspending vehicle can be heated and the granular formulation added to the suspending vehicle under anhydrous conditions. The ingredients can be mixed under vacuum at elevated temperatures, such as from about 40°C to about 70°C. The ingredients may be mixed at a speed sufficient (such as from about 40 rpm to about 120 rpm) and for a sufficient time (such as about 15 minutes) to achieve uniform dispersion of the particulate formulation in the suspending vehicle. The mixer may be a double helix blade or other suitable mixer. The resulting mixture can be removed from the mixer, sealed in a dry container to prevent water contamination of the suspended formulation, and allowed to cool to room temperature before further use, such as filling into an implantable drug delivery device, unit dose container, or multiple Dosing container.

Suspension formulations generally have an overall moisture content of less than about 10 wt%, preferably less than about 5 wt%, and more preferably less than about 4 wt%.

In preferred embodiments, the suspension formulations of the present invention are substantially homogeneous and flowable to provide delivery of the drug particulate formulation from an osmotic delivery device to an individual.

Briefly, the components of the suspension vehicle provide biocompatibility. The components of the suspending vehicle provide suitable chemical-physical properties to form a stable suspension of the drug particulate formulation. These properties include, but are not limited to, the following: viscosity of the suspension; purity of the vehicle; residual moisture in the vehicle; density of the vehicle; compatibility with anhydrous powders; compatibility with implantable devices; polymer properties Molecular weight; stability of the vehicle; and hydrophobicity and hydrophilicity of the vehicle. These properties can be adjusted and controlled, for example, by changing the vehicle composition and adjusting the ratios of the components used in the suspending vehicle.

4.0.0 Delivery Suspension Formula

The suspension formulations described herein can be used in implantable osmotic delivery devices to provide zero-order, continuous, controlled and Continuous delivery. The implantable osmotic delivery device can generally deliver a suspension formulation containing the drug at a desired flow rate and for a desired period of time. The suspended formulation can be loaded into an implantable osmotic delivery device using conventional techniques.

The dosage and delivery rate can be selected to achieve a desired blood concentration of the drug in substantially less than about 6 drug half-lives after implantation of the device in the individual. Drug blood concentrations are selected to give the optimal therapeutic effect of the drug while avoiding undesirable adverse effects that can be induced by excessive drug concentrations and avoiding spikes and troughs that can induce adverse effects associated with peak or trough plasma concentrations of the drug.

Implantable osmotic delivery devices generally include a reservoir having at least one aperture through which the suspended formulation is delivered. Suspension formulas can be stored in storage tanks. In a preferred embodiment, the implantable drug delivery device is an osmotic delivery device, wherein the delivery of the drug is osmotically driven. Several osmotic delivery devices and components thereof have been described, such as the DUROS® delivery device or similar devices (see, for example, U.S. Patents 5,609,885; 5,728,396; 5,985,305; 5,997,527; 6,113,938; 6,132,420; 6,156,331; 6,217,906; 6,261,584; 6,2 70,787; 6,287,295; 6,375,978; 6,395,292; 6,508,808; 6,544,252; 6,635,268; 6,682,522; 6,923,800; 6,939,556; 6,976,981; 6,997,922; 7,014,636; 7,207,982; and 7,112,335; 7,163,688; U.S. Patent Publication Nos. 2005/0175701, 2007/0281024, 2008/0091176 and 2009/02 02608).

Osmotic delivery devices generally consist of a cylindrical reservoir containing an osmotic engine, a piston and a drug formula. One end of the reservoir is capped by a rate-controlled semipermeable membrane, and the other end is capped by a diffusion regulator through which the suspended formulation containing the drug is released from the drug reservoir. The piston separates the drug formula from the osmotic engine, and a seal is used to prevent water in the osmotic engine compartment from entering the drug reservoir. The diffusion adjuster is designed with the drug formula to prevent body fluids from entering the drug storage tank through the holes.

Osmotic devices release drugs at a predetermined rate based on the principle of osmosis. Extracellular fluid passes directly through the semipermeable membrane into the salt engine of the osmotic delivery device, which expands at a slow and consistent delivery rate to drive the piston. The piston motion forces the drug formulation to be released through the orifice or exit port at a predetermined shear rate. In one embodiment of the invention, a reservoir of an osmosis device is filled with a suspended formulation, wherein the device is capable of operating for an extended period of time (e.g., about 1, about 3, about 6, about 9, about 10, and about 12 months) The suspension formulation is delivered to the subject at a predetermined, therapeutically effective delivery rate.

The drug release rate of an osmotic delivery device generally provides a predetermined target dose of drug to an individual, such as delivering a therapeutically effective daily dose over the course of a day; that is, the drug release rate of the device provides substantially steady-state delivery of a therapeutic concentration of drug to an individual. individual.

Generally speaking, for osmotic delivery devices, it is beneficial to include The benefit agent chamber volume of the agent formulation is between about 100 μl and about 1000 μl, more preferably between about 120 μl and about 500 μl, and more preferably between about 150 μl and about 200 μl.

Typically, osmotic delivery devices are implanted within an individual, such as subdermally or subcutaneously, to provide subcutaneous drug delivery. The device may be implanted subdermally or subcutaneously in either arm or both arms (eg, inside, outside, or behind the upper arm) or in the abdomen. The best place to tie in the abdominal area is under the abdominal skin, extending from below the ribs to just above the waistline. To provide some abdominal sites for implantation of one or more osmotic delivery devices, the abdominal wall can be divided into 4 quadrants as follows: The upper right quadrant extends at least 2 to 3 cm below the right rib (e.g., at least about 5 to 8 cm below the right rib) And at least 2 to 3 cm to the right of the midline (for example, at least about 5 to 8 cm to the right of the midline); the lower right quadrant extends at least 2 to 3 cm above the waist line (for example, at least about 5 to 8 cm above the waist line) and at least 2 to 3 cm to the right of the midline (for example, at least about 5 to 8 cm to the right of the midline); the upper left quadrant extends at least 2 to 3 cm below the left rib cage (for example, at least about 5 to 8 cm below the left rib cage) and At least 2 to 3 cm to the left of the midline (e.g. at least about 5 to 8 cm to the left of the midline); and the lower left quadrant extends at least 2 to 3 cm above the waist line (e.g. at least about 5 to 8 cm above the waist line) and At least 2 to 3 cm to the left of the midline (for example, at least about 5 to 8 cm to the left of the midline). This provides multiple sites for one or more implants of one or more devices. Implantation and removal of the osmotic delivery device is typically performed by a medical professional using a local anesthetic (eg, lidocaine).

Terminating treatment by removing the individual's osmotic delivery device is straightforward and provides the important advantage of immediately ceasing drug delivery to the individual.

Preferably, the osmotic delivery device has a fail-safe mechanism to prevent accidental overdose or bolus delivery of the drug under theoretical conditions such as plugging or clogging of the outlet (diffusion regulator) of the delivered drug formulation. To prevent accidental overdose or bolus delivery of drugs, osmotic delivery devices are designed and constructed such that the pressure required to partially or completely relieve or detach the diffusion regulator from the reservoir exceeds the pressure required to partially or completely detach or detach the semipermeable membrane to The pressure required to the extent that the tank needs to be depressurized. In this case, pressure will build up within the device until it pushes the semipermeable membrane at the other end outward, thereby releasing the osmotic pressure. The osmotic delivery device will then become stationary and no longer deliver the drug formulation, but the piston will remain in a sealed relationship with the reservoir.

Suspension formulas can also be used in infusion pumps, such as the ALZET® (DURECT Corporation, Cupertino, Calif.) osmotic pump, which is a small infusion pump used for continuous dosing of experimental animals (such as mice and rats).

Example

The following examples are set forth to provide a complete disclosure and explanation of how to practice the invention to those of ordinary skill in the art, and are not intended to limit the scope of the inventor's claims for the invention. Every effort has been made to ensure the accuracy of the numbers used (such as amounts, concentrations, and percentage changes), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, temperatures are in degrees Celsius and pressures are at or near atmospheric pressure.

The composition used to implement the method of the present invention meets the content and purity specifications specified for pharmaceutical products.

Example 1: Production of glucagon receptor selective agonist polypeptides

The glucagon polypeptides of the present invention (as provided in Table 3) were synthesized by a solid-phase method on a Prelude peptide synthesizer (Protein Technologies Inc., Tucson, AZ) using an Fmoc strategy with 2-(1H-benzo Triazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (HBTU) or 2-(6-chloro-1-H-benzotriazol-1-yl)-1 , 1,3,3-tetramethylammonium hexafluorophosphate (HCTU) (5-fold molar excess) was activated in N,N-dimethylformamide (DMF) and N'N- Diisopropylethylamine (DIEA) was used as base and 20% piperidine/DMF was used for Fmoc deprotection. The resin was Rink Amide MBHA LL (Novabiochem) or N-α-Fmoc protected, preloaded Wang LL (Novabiochem), loaded at 0.29 to 0.35 mmol/g in the 20 to 400 μmol scale. The resin was treated with (92.5% TFA, 2.5% phenol, 2.5% water, and 2.5% triisopropylsilane) for 2 to 3 hours to allow for final deprotection and free-standing cleavage of the peptide. The cleaved peptides were precipitated using cold diethyl ether. The diethyl ether was decanted and the solid was triturated again with cold diethyl ether, centrifuged into a pellet, and freeze-dried. The freeze-dried solid was redissolved in 1:1 acetonitrile/water (10 to 15 mL) containing 0.1% TFA and analyzed via reverse phase HPLC on a Waters XBridge ^™ BEH 130, CIS, 10pm, 130Å, 30×250mm ID column. Purification was performed using a 30 gradient in the range of 5 to 75% acetonitrile/water (containing 0.1% TFA) over 30 to 45 minutes, flow rate 30 mL/min, λ-215 nm. The purified product was freeze-dried and analyzed by ESI-LC/MS and analytical HPLC; the results showed pure product (>98%). The quality results are consistent with the calculated values.

Peptide analogues were characterized by C18 HPLC and LC/MS analysis (Acquity SQD Waters Corp, Milford, MA) and UV detection provided by dual absorption signals at 215 nm and 280 nm, using three methods (A or B or C ) one of them.

LC/MS conditions: Method A: Use Phenomenex UPLC Aeris ^TM Peptide /water (containing 0.05% TFA) for 30 minutes, flow rate 0.5mL/min, λ-215nm, 280nm.

C18 HPLC conditions: Method A: UPLC analysis system on Acquity BEH130, C18 column, 1.7μm, 100×2.10mm column at 25℃, 5 to 65% acetonitrile/water (containing 0.05% TFA) for 30 minutes , flow rate 0.5mL/min, λ 215nm, λ 280nm.

Method B: UPLC analysis system on Acquity BEH130, C18 column, 1.7μm, 100×2.10mm column at 25°C, with 5 to 65% acetonitrile/water (containing 0.05% TFA) for 20 minutes, flow rate 0.5mL /min, λ 215nm, λ 280nm.

Method C: UPLC analysis system on Acquity BEH130, C18 column, 1.7μm, 100×2.10mm column at 25℃, with 5 to 65% acetonitrile/water (containing 0.05% TFA) for 10 minutes, flow rate 0.5mL /min, λ 215nm, λ 280nm. A 5.0uL sample was injected using PLNO (partial loop plus needle overflow) injection mode.

Table 3 provides the amino acid sequences and experimental data of selective glucagon receptor analogs of the present disclosure, including compounds referred to herein as A4 (SEQ ID NO: 44), compound A5 (SEQ ID NO: 45), compound A6 (SEQ ID NO: 46), compound A2 (SEQ ID NO: 42), compound A1 (SEQ ID NO: 41), compound A3 (SEQ ID NO: 43), compound A97 (SEQ ID NO: 143), compound A98 (SEQ ID NO: 144), compound A99 (SEQ ID NO: 145), compound A100 (SEQ ID NO: 146), compound Selective glucagon receptor analogs of A101 (SEQ ID NO: 147), compound A102 (SEQ ID NO: 148), and compound A97 (SEQ ID NO: 149).

Example 2: Production of lactamine-bridged glucagon receptor selective agonist polypeptides

Synthesis, purification and analysis were performed as described in Example 1 with the following modifications. The glucagon polypeptides of the present invention (as provided in Table 3) were synthesized by solid-phase methods on a Prelude peptide synthesizer (Protein Technologies Inc., Tucson, AZ) using the Fmoc strategy and using Rink Amide MBHA LL (Novabiochem ) or N-α-Fmoc protected, preloaded Wang LL (Novabiochem) at 0.29 to 0.35 mmol/g loading in the 20 to 400 μmol scale. Fmoc amino acid (4.0eq, 1.0mmol) residues were activated using 4.0eq HBTU, 4.0eq HOBT, 8.0eq DIEA and coupled to the resin for 1 hour. The Fmoc group was removed by treatment with dimethylformamide containing 20% (v/v) piperidine. The side chain protecting groups used are Trt for Asn, Gln, Cys, and His; t-Bu for Ser, Thr, and Tyr; Boc for Lys and Trp; Ot-Bu for Asp and Glu; and Pbf for Arg.

The lactam bridge between positions 17 to 21 and 24 to 27 was orthogonally protected using lysine (allyloxycarbonyl) 17 and glutamic acid (allyl) 21 is introduced; and glutamic acid (allyl) 24 and lysine acid (allyloxycarbonyl) 27 are represented by E in brackets in the sequence, lysine acid (allyloxycarbonyl) These are represented by K in parentheses in the sequences SEQ ID NO: 19 and SEQ ID NO: 20.

The synthesis is carried out on the support from the C-terminus to the N-terminus. The synthesis was paused at Phe21 for incorporation of the first lactam bridge (residues 17 to 21). The resin was washed with DCM (6 x 10 ml), which had been previously flushed with nitrogen for 30 minutes. Next, tetrakis(triphenylphosphine)palladium (Pd(PPh3)3) (3 equiv) was added to the CHCl3/AcOH/NMM (37:2:1) solution. Bubble nitrogen through the solution until all solids dissolve, leaving a dark amber solution. This solution was transferred to an amino acid bottle where it was degassed with nitrogen for an additional 5 minutes. Then place it on the Prelude synth. 20 ml of palladium solution was added to each RV and the reaction mixture was allowed to stir for 3 hours. Synthesis was continued using standard protocols. To incorporate the second lactam bridge (residue 24 to residue 28), the synthesis was paused at Leu14 and the lactam bridge formation procedure described above was repeated.

Before cleaving the peptide from the resin, the allyl protecting group was removed using Pd(PPh3) ₃ in _CHCl3 /AcOH/NMM (37:2:1). Cyclization to lactam was performed using Pybop (6 equiv.), HOBT (6 equiv.), and DIEA (12 equiv.). The resin was then washed with DMF and dried under nitrogen under vacuum for 15 minutes.

The resin was treated with Reagent B (92.5% TFA, 2.5% phenol, 2.5% water, and 2.5% triisopropylsilane) for 2 to 3 hours to allow for final deprotection and free-standing cleavage of the peptide. The cleaved peptide was precipitated using cold diethyl ether, the diethyl ether was decanted, and the solid was triturated again with cold diethyl ether, centrifuged into a pellet, and freeze-dried. The pellet was redissolved in water (10 to 15 mL), ^filtered , and analyzed by reverse phase HPLC on a Waters A 30 gradient in the water range (containing 0.1% TFA) was purified at λ-215nm over 30 to 45 minutes, flow rate 30mL/min. Alternatively, purification was performed via reverse phase chromatography using a Gilson preparative HPLC system using a Waters XBridge BEH 130, C18, 10 μm, 130 Å, 30 × 250 mm ID column with a gradient ranging from 5 to 45% acetonitrile/water containing 0.1 % TFA) over 30 to 45 minutes, flow rate 30 mL/min, λ 215 nm. The purified product was freeze-dried and analyzed by ESI-LC/MS and analytical HPLC; the results showed pure product (>98%). The quality results are consistent with the calculated values.

Peptide analogues were characterized by C18 HPLC and LC/MS analysis (Acquity SQD Waters Corp, Milford, MA) and UV detection provided by dual absorption signals at 215 nm and 280 nm, using three methods (A or B or C ) one of them.

The chemical structure of compound A104 is shown in Figure 4A, and the chemical structure of compound A105 is shown in Figure 4B.

Example 3: Stability and solubility of glucagon receptor selective agonist polypeptide

Stability of glucagon and analogs described herein, such as Compound A98, Compound A99, Compound A100, Compound A101, Compound A102, Compound A2, Compound A1, Compound A5, Compound A6, Compound A3, Compound A44, and Compound A97 Measured in saline 5% NaCl in water (i.e., bioassay buffer) or in water (i.e., in water) Try. Glucagon and these analogs were cultured at 37°C and at room temperature. Samples were taken at specified intervals for 30 days and analyzed by LC/MS and HPLC to determine the purity and quality of the degradation products. The results of this analysis are shown in Table 3.

The solubility system of glucagon and analogs described herein, such as Compound A98, Compound A99, Compound A100, Compound A101, Compound A102, Compound A2, Compound A1, Compound A5, Compound A6, Compound A3, Compound A44, and Compound A97 Test in saline 5% NaCl in water (i.e., bioassay buffer) or in water at room temperature. Visually inspect the sample for clarity and any signs of turbidity or haze. The results of this analysis are shown in Table 3.

Example 4: In vitro screening of analogues for peptide-receptor-mediated cAMP accumulation

Using Lipofectamine 2000, CMV promoter-driven plasmids encoding human GCGR (NM_000160) and human GLP-1R (NM_002062) were transfected into CHO-K1 cells for 36 hours. After transfection, cells were removed from the flask using cell disruption solution and distributed into a white 384-well plate at 1000 cells per well in 5uL stimulation buffer. Peptide-receptor activity was determined using the LANCE Ultra cAMP detection kit according to the manufacturer's protocol (Perkin Elmer). The peptides were serially diluted with stimulation buffer and 5uL of each peptide dilution was added to the cells and incubated at room temperature for thirty minutes. For the results shown in Table 2A, the test concentration range was from 1 nM to 15 fM in the GCGR and GLP-1R assays. For the results shown in Table 2B, the test concentration range was from 1 uM to 0.1 fM in the GCGR assay and 500 uM to 10 fM in the GLP-1R assay. After peptide incubation, 5 uL Europium-labeled cAMP and 5 uL Ulight ^™ anti-cAMP antibody were added to the wells and incubated for an additional 60 minutes. Plates were read on an Envision® fluorescent plate reader and data analyzed using GraphPad Prism®. Potency is determined from the baseline correction fitting curve, using the formula: Y=Bottom+(Top-Bottom)/(1+10^((LogIC50-X)*HillSlope)), where Hill Slope is limited to -1.0.

Human glucagon, GLP-1(7-37), and two glucagon receptor-selective peptide agonists for human glucagon receptor (GCGR) and human GLP-1 receptor (GLP-1R) Peptide-receptor activation profile. Figure 1A shows that Compound A2, the peptide of Compound Al, and glucagon are almost equally potent on GCGR, whereas GLP-1(7-37) activates GCGR with much lower potency. Figure 1B shows the phase Regarding the results shown in Figure 1A, peptide compound A2 and compound A1 are inactive towards GLP-1R, indicating that peptide compound A2 and compound A1 function as GCGR selective agonists. Information for all analogues is recorded in Table 3.

The sequences of the synthetic glucagon receptor agonist peptides and their pEC50 values measured in the cAMP assay are shown in Table 3 below.

Example 5: Intravenous Infusion: Pharmacokinetic Test to Evaluate Renal Clearance (CL) of Peptides

The peptide was prepared in sterile saline and administered via a jugular vein cannula to non-fasted male Wistar Han or Sprague-Dawley rats (n=3 per group) as a 3-hour intravenous infusion at a final dose of 0.3 or 0.1 mg/kg. The formula is administered at a rate of 1.67 mL/kg/h. Blood samples (approximately 250 uL) were collected for pharmacokinetic analysis via a femoral vein cannula at 1, 2, 3, 3.17, 3.33, 3.5, 4, 4.5, 5, and 6 hours after the start of infusion to contain K2EDTA as Anticoagulant and 25uL of protease inhibitor cocktail in a microblood collection tube. Plasma was prepared by centrifugation and stored at -80°C until analysis.

Example 6: Subcutaneous Infusion: Pharmacokinetic Test to Evaluate Renal Clearance (CL) of Peptides

The peptide was prepared in sterile saline and administered as a 3-hour subcutaneous infusion via a cannula placed in the subcutaneous space between the shoulder blades into non-fasted male Wistar Han or Sprague-Dawley rats (n=3 per group). The final dose is 0.3 or 0.1 mg/kg. The formula is administered at a rate of 0.145mL/kg/h. Blood samples (approximately 250 uL) were collected for pharmacokinetic analysis via jugular vein cannula at 1, 2, 3, 3.17, 3.33, 3.5, 4, 4.5, 5, and 6 hours after the start of infusion to contain K2EDTA as Anticoagulant and 25uL of protease inhibitor cocktail in a microblood collection tube. Plasma was prepared by centrifugation and stored at -80°C until analysis. The results of this analysis are shown in Table 3 below and Figures 2A and 2B.

Example 7: Intravenous bolus injection of a cyclic peptide glucagon analog: a pharmacokinetic test to assess renal clearance (CL) of the peptide

The peptide was administered as a single intravenous bolus dose via jugular vein cannula to non-fasted male Wistar Han rats (n=3 per group). Compounds were prepared as solutions in sterile saline, acidified saline (pH 2.0 or 4.5), or water containing 5% DMSO and administered at a volume of 1.5 mL/kg and a final dose of 0.1 mg/kg. Blood samples (approximately 250uL) were collected for pharmacokinetic analysis. They were collected through femoral vein cannula at 0.083, 0.167, 0.25, 0.33, 0.5, 1, 2, 4, 8, 12, and 24 hours after administration to contain K2EDTA is used as an anticoagulant and 25uL of protease inhibitor cocktail in microblood collection tubes. Plasma was prepared by centrifugation and stored at -80°C until analysis. The results of this analysis are shown in Table 4 below.

Example 8: General method for preparing plasma samples for pharmacokinetic testing

Protein precipitation: All 96-well plates are coated with blocking agent to mitigate non-specific binding of peptides. Plasma samples (75uL) were added to a 96-well plate containing 200uL of 2:1 ethanol:acetonitrile (containing 0.1% TFA) and Mix evenly by pumping. The plate was capped, vortexed, and centrifuged. The supernatant (215 μL) was transferred to a clean 96-well plate, evaporated to dryness under a stream of nitrogen, and then reconstituted in 75 μL of 20% acetonitrile in water (containing 0.1% formic acid).

Solid phase extraction: The sample was diluted 3-fold with 5% NH ₄ OH (aq) and loaded onto an Oasis MAX microElution plate (Waters Corp, Milford, MA), which had been incubated with 200uL each of methanol and 5% NH ₄ OH. (aq) Preconditioning. The plate was washed with 200uL 5% _NH4OH (aq) followed by 200uL 20% acetonitrile in water. The peptide was eluted with 200 uL of methanol containing 5% formic acid and evaporated under a stream of nitrogen. The sample was reconstituted with 80uL of water containing 0.1% formic acid.

Example 9: LC/MS quantification of peptides in plasma

All calibration standards were prepared in control rat plasma containing K2EDTA and protease inhibitor cocktail. Samples and standards were analyzed by TurboIonSpray ^TM UPLC-MS/MS using a CTC HTS PAL autoinjector (Leap, Carrboro, NC), an Agilent Infinity 1290 system with column oven (Palo Alto, CA), and a Valco switching valve. (Houston, TX), and a system composed of AB Sciex API 5600 TripleTOF ^TM or Sciex API 4000QTrap mass spectrometer (Framingham, MA). The sample is injected onto a 2.1×50 mm reverse phase C18 analytical column, typically a Waters ACQUITY UPLC ^™ HSS T3, 1.8 μm (Waters Corporation, Milford, MA) or similar column. Chromatographic separation was achieved by a gradient method, using water (A) containing 0.1% formic acid and acetonitrile (B) containing 0.1% formic acid as mobile phases. The initial conditions consist of 95% A and 5% B. Increase the organic component linearly to 95% B over 3 to 4 minutes (depending on the peptide). The general flow rate is 600μL/min. Keep the column temperature constant at 40 or 45°C. Quantification of peptides is performed by monitoring one or more product ions produced from multiply charged precursor ions.

Example 10: In vivo efficacy of glucagon analogues in weight loss

Chronic (13 days) in vivo efficacy trials were conducted in a rodent obesity model (diet-induced obesity (DIO) Long Evans rats) to investigate the efficacy of Examples 11 and 12 alone and in combination with secretagogue-4 as anti-obesity agents and Persistence.

Male diet-induced obesity (DIO) Long Evans (LE) rats (Harlan Laboratories, Inc., Indianapolis, IN) were used and fed a high-fat diet (Teklad TD 95217, 40% kcal) starting from the time of weaning (approximately 3 weeks of age). from fat, Harlan Laboratories, Madison, WI) fed to rats. At the start of the experiment, the rats were 15 to 17 weeks old. Rats were housed in one cage per cage, with access to high-fat feed (Harlan TD.95217, 4.3kcal/g) and water ad libitum, and maintained on a 12hr light/dark cycle from 5:00 AM to 5:00 PM. 21°C, relative humidity 50%, and allowed to acclimate for at least 10 days before surgery. Baseline fat mass and fat-free mass measurements were taken 3 days before starting peptide infusion using a QMR instrument (Echo Medical Systems, Houston, TX). Body weight measurements were taken twice a week starting three days before surgery. Rats were randomly divided into treatment groups according to their body fat mass and/or body weight (n=4 to 6 rats/group). Alzet small osmotic pump (2 weeks; Model 2002, Durect Corporation, Cupertino, CA) was administered the day before surgery. Fill the vehicle or peptide under sterile conditions. On the day of surgery, rats were anesthetized with isoflurane, and the dorsal skin surface was shaved and cleaned. Rats were injected SC with Flunexin (2.5 mg/kg). A 1 to 2 cm surgical incision is made between the shoulder blades. Using a blunt instrument, create a 2 to 3 cm subcutaneous channel into which a sterile, filled, small osmotic pump is placed. The skin opening is closed with skin staples. Depending on the rat's treatment group, each rat was implanted with one or two osmotic pumps containing vehicle or peptide. All data are expressed as mean ± SEM. Data were analyzed in Excel and/or Prism (GraphPad Software, Inc., La Jolla, CA) using one-way ANOVA to compare each group to appropriate controls. P-values <0.05 were considered to indicate significant differences between treatment groups.

Animals were raised and maintained under an AAALAC International accredited care and use program. All procedures were performed in accordance with the U.S. Department of Agriculture Animal Welfare Act and were approved by the GlaxoSmithKline or Mispro Laboratory Animal Care and Use Committee.

Example 11: Efficacy: Body weight changes in DIO rats after 13 days

In DIO LE rats, continuous administration of Compound A2 and Compound A1 for 13 days resulted in dose-dependent weight loss. Compared with the vehicle control, compound A2 at doses of 0.01, 0.03, 0.1 and 0.3 mg/kg/day achieved significant efficacy in weight loss of 3.7, 3.9, 8.6 and 23.7%, respectively (p<0.05) (Figure 3B). However, compared to the vehicle control, Compound A1 at doses of 0.01, 0.03, 0.1 and 0.3 mg/kg/day achieved weight loss of 5.6, 3.5, 10 and 22.6%, respectively (p<0.05) (Figure 3C).

Example 12: The efficacy of compound A104 alone or in combination with secretagogue-4 in DIO LE rats

Continuous administration of Compound A104 to DIO LE rats for 13 days resulted in dose-dependent weight loss. Compared to the vehicle control, Compound A104 at doses of 0.1, 0.3 and 1.0 mg/kg/day achieved significant efficacy in weight loss of 4.2%, 16.5% and 25.2%, respectively (p<0.05) (Figure 3A, left panel). Next, a combination of a cyclic peptide glucagon analog and insulin secretin-4 is used. This polypeptide contains the amino acid sequence H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser. -Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro -Pro-Pro-Ser- _NH2 (SEQ ID NO: 142). Compound A104 at doses of 0.03, 0.1, 0.3, and 1 mg/kg/d compared to vehicle control at 13 After days, 6.7, 11.7, 24.3 and 29.7% body weight loss was achieved in DIO LE rats (p<0.05) (Figure 3A, right panel).

Example 13: Steady-state plasma concentrations and intravenous infusion rates of exenatide and GLP-1

Methods: Referring to the chart in Figure 5, male Sprague-Dawley rats (Harlan, Indianapolis, IN) weighing 350 to 370 g were allowed to eat water and food (Diet LM-485, Teklad, Madison, WI) ad libitum until the experiment. 18 hours before. During catheterization of the saphenous vein (for peptide infusion) and right femoral artery (for blood collection and monitoring of arterial blood pressure), the animals were Halogen anesthesia. Measure and control colonic temperature. Exenatide or GLP-1-(7-36)amide was continuously infused for 3 hours at one of three infusion rates of 0.5, 5, or 50 nmol/h (n=4 to 6 in each group). Arterial samples were collected every 30 min into heparinized Natelson capillary tubes, plasma was separated in a tabletop centrifuge, and then frozen at -20°C until assayed. Protease inhibitors were added to blood samples collected for GLP-1 measurement, and assayed using Linco, Kit No. EGLP-35K. The assay of exenatide uses a two-site sandwich assay.

Results: During infusions of 0.05, 0.5, 5, and 50 nmol/h, plasma concentrations of secretin-4 and GLP-1 reached steady state in approximately 30 minutes. The steady-state plasma concentrations of the two peptides were each dependent on the infusion rate. The relationship between infusion rate and steady-state plasma concentration (average over the final 2 hours) is shown as the three data points furthest below the GFR, and the relationship for exenatide is shown as the three data points closest to the GFR (should be Note that both the X-axis and Y-axis are in logarithmic units). These relationships can be used to calculate clearance for each dose. The plasma clearance of exenatide ranges from 3.7±0.5 to 8.3±0.7mL/min. The clearance of GLP-1 ranged from 34±4 to 38±3mL/min, and was approximately 12 times higher than that of exenatide. The clearance of exenatide is similar to that reported for inulin (a marker of glomerular filtration rate). The relationship between the expected steady-state concentration of inulin (cleared only by glomerular filtration, GFR) and the infusion rate is shown as a dashed black line.

Example 14: Steady-state plasma concentrations and intravenous infusion rates of selected glucagon analogues

Referring to the graph of Figure 6, these data include the data shown in the graph of Figure 5 and additionally include the data when native glucagon was infused intravenously into anesthetized rats (using the procedure described in Example 13) at 2 different infusion rates. Steady state concentration (inverted triangle). The square data point closest to the GFR represents the plasma concentration measured when 2 glucagon analogs (compounds A1 and A2) were administered continuously into the subcutaneous space via an osmotic minipump. Square data points close to the dotted GFR line indicate (a) high subcutaneous bioavailability and (b) clearance from the vascular compartment consistent with glomerular filtration.

Example 15: Clearance value of selected glucagon analogues

Referring to the illustration in Figure 7, clearance was determined in multiple experiments (eg 14) performed as above, but the intravenous infusion rate was uniformly 10mcg/kg/hr. Selected clearance values (expressed in mL/min per kg body weight) obtained in these experiments are provided in Table 5 below.

The three dark spots distributed above and closest to the GFR are from columns 1, 2, and 5 in Table 5, corresponding to compounds A1, A2, and A3. Repeating the experiments in columns 3 and 4 yields values very similar to those in columns 1 and 2. Note that the vertical axis is logarithmic and inverted so that the ligand with the lowest clearance value is at the top of the scatter plot. The solid line labeled GFR represents published values for rats. from column Values of 1, 2, and 5 are close to this boundary, consistent with clearance of these analogs from plasma via glomerular filtration. The other dark spots on the dotted line labeled "Glucagon" furthest from the GFR represent the values obtained for native (human/rat) glucagon in this assay. The other dashed line closest to the GFR, labeled "exenatide", is derived from the published values obtained for exenatide in this model system. The clearance of exenatide is similar to glomerular filtration, but a small portion is cleared by means other than glomerular filtration. Several analogues are closer to glomerular filtration than exenatoid, which is their limited clearance mode. Without being bound by theory, these analogs may be more resistant to peptidase digestion than exenatide. Several analogs have higher clearance than native glucagon, showing that all changes in the sequence are not necessarily beneficial.

Example 16: Comparison of dose-dependent weight loss in rats following continuous administration of exenatide or selected glucagon analogs

Referring to the graph of Figure 8, the black circles are a plot of the average change in body weight expressed as a percentage of initial body weight in rats with diet-induced obesity 13 days after implantation of a small pump delivering Compound A1 (eg, as described in 10). Symbols are mean±SEM (n=4/dose group). Rates of delivery to the subcutaneous space were 10, 30, 100, 200, 300 and 1000mcg/kg/day. Percent change in body weight is relative to the vehicle-infused group ("zero" on the x-axis). The curve based on the circle of data points best fits a 4-parameter sigmoid function, limiting the vehicle response to 0, and the maximum response to -28%. The derived ED50 is 65.7mcg/kg/day. Hill slope is -1.26.

Data for exenatide tested in the same model are shown as Inverted triangle. Exenatide infusion rates were 3, 10, 40, 100, 150, and 500 mcg/kg/day. The resulting curve based on the triangle of data points best fits a 4-parameter sigmoid function, limiting the mediator response to 0. The derived maximum response was -12.2% weight loss. The ED50 is 10.4mcg/kg/day and the Hill slope is -1.85.

As shown in the data in Table 6 below, in vivo administration of Compound A1 alone was nearly as effective as exenatide and resulted in 2.3-fold greater maximum weight loss than exenatide.

Example 17: Comparison of dose-dependent weight loss in rats following continuous administration of selected glucagon analogues

Referring to the diagram in Figure 9, the symbols represent diet-induced obese rats infused with Compound A1 (circles), Compound A2 (squares) or a combination of The mean ± SEM of the percentage change in body weight of object A3 (triangle) after 13 days (as shown in Figure 8). The infusion rates of Compound A1 were 0, 10, 30, 100, 200, 300 and 1000mcg/kg/day. Compound A2 was 0, 3, 10, 30, 100, 200 and 300mcg/kg/day and Compound A3 was 0, 3, 10, 30, 100 and 300mcg/kg/day.

The three curves represent the best-fitting 4-parameter S-shaped curve for each data set. Fits were restricted to sharing the same percent change in body weight as the vehicle group. Since all act through a common (glucagon signaling) pathway, they are limited to a common maximum weight loss response, which was derived to 27.2% after 13 days. For compounds A1, A2 and A3, the ED50s were 51, 271 and 535mcg/kg/day respectively. The Hill slopes are -1.43, -1.25 and -1.12 respectively. Experiments show that even if each analog has been optimized for pharmacokinetic properties, in vivo potency still varies, and experimentation is needed to select the analog with the highest in vivo potency.

Example 18: Comparison of dose-dependent weight loss in rats following continuous administration of selected glucagon analogues alone or in combination with exenatide

The data on the graph of Figure 10 represents the mean (± SEM) percentage change in body weight of rats with diet-induced obesity after 27 days of treatment. The average initial body weight was 564±4g. Animals (n=8/dose group) were implanted with an osmotic minipump to deliver Compound A1 with or without co-delivery of exenatide 10 mcg/kg/day (eg, as described in 10). Compound Al was delivered at a rate of 0, 7.5, 24 or 50mcg/kg/day. Therefore, there are two dose-response experiments of compound A1, one without exenatide and one with exenatide. Peptides. There is also a "pair feeding" group, which provides the same amount of food consumed on the previous day as the compound A1 50mcg/kg/day + exenatide 10mcg/kg/day treatment group. The purpose of the pair-fed group was to estimate the amount of weight loss in the latter group that could be attributed to reduced caloric intake compared to other mechanisms.

Black circles plot 27-day body weight changes in animals treated with Compound Al (or vehicle) alone. Animals infused with vehicle or Compound A1 at 7.5 and 24 mcg/kg/day showed a small increase in body weight of approximately 4%, however the highest infusion rate showed a decrease of 16.8%.

Triangles represent the 27-day percent change in body weight for a similarly treated group but with the addition of exenatide 10 mcg/kg/day. The infusion itself resulted in a 4.2% body weight loss, a relative difference of 7.9% in the vehicle-only group. The squares represent the expected 7.9% reduction in exenatide alone plus the observed weight change in the Compound A1 only group, representing the arithmetic sum of the responses of each component.

The dashed line is the fitted S-shaped curve of the arithmetic summation response obtained by this method. The observed response for the combination of the 2 highest infusion rates of Compound A1 (24 and 50 mcg/kg/day) with exenatide gave greater than expected body weight relative to the expected response (which is the arithmetic sum of the responses for the single doses) alleviate. In other words, there is a super bonus (synergistic) effect. The maximum weight loss response was not about 27%, but about 47%.

Example 19: Effect of glucagon analogues on weight loss in DIO LE rats after 27 days

Using the procedure described above, monitor DIO LE rats after administration of exacerbation after exenatide, a glucagon (“GCG”) analog (derived ED10, ED30 and ED50 values), or a combination of exenatide and a GCG analog (derived ED10, ED30 and ED50 values) weight loss. The percentage of weight loss 27 days after treatment was calculated (Figure 11). Figure 11 is a bar graph alternatively depicting the data shown in Figure 10.

Example 20: Effects of glucagon analogues on serum triglycerides, liver fat content and liver weight in Zucker diabetic fatty liver (ZDF) rats after 14 days

Six-week-old male Zucker diabetic fatty liver (ZDF) rats (Charles River, Raleigh, NC) were obtained and used for experiments at 8 weeks of age. Upon receipt, rats were housed in one cage per cage using α dri bedding (Shepherd Specialty Papers, Inc., Kalamazoo, MI), with ad libitum access to Purina 5008 feed (Lab Diet, St. Louis, MO) and Water, maintain a 12hr light/dark cycle from 5:00 AM to 5:00 PM, a temperature of 21°C, and a relative humidity of 50%, and allow to adapt for 9 days before starting the test.

Rats were administered exenatide (0.01 mg/kg/day in 10% DMSO/water) and/or glucagon analog via subcutaneous (s.c.) placement of an Alzet Osmotic Pump Model 2002 (DURECT Corporation, Cupertino, CA). substance, compound A104 (0.03, 0.1, 0.3, 1.0 or 3.0 mg/kg/day in 10% DMSO/water). Alzet pumps were aseptically filled with vehicle (10% DMSO/sterile water) or exenatide on the day of surgery. The rats were anesthetized with isoflurane, and the dorsal skin surface was shaved and cleaned with lohexidine and sterile water. Rats were injected ID with Lidocaine (an analgesic, 0.1 ml of 0.125% Lidocaine). A 1 to 2 cm surgical incision is made between the shoulder blades. Using a blunt instrument, create a 2 to 3 cm subcutaneous channel into which a sterile, filled, small osmotic pump is placed. The skin opening is closed with skin staples. Rats were monitored for recovery from isoflurane anesthesia after surgery.

Baseline blood samples (Day -3) were collected via the tail vein to measure triglyceride levels. Final blood samples were collected by cardiac puncture under isoflurane anesthesia (day 14). Sera were prepared according to manufacturer's protocols using EDTA and T-MG tubes [Terumo Medical Corporation, Elkton, MD], respectively. After terminal blood collection, livers were collected, weighed and homogenized for biochemical analysis (Figure 13). Clinical chemistry analysis of serum samples and liver homogenates was performed using an Olympus AU640 Clinical Chemistry analyzer (Olympus America Inc., Melville, NY), following the manufacturer's protocols and method parameters for triglyceride determination.

Animals were raised and maintained under an AAALAC International accredited care and use program. All procedures were performed in accordance with the U.S. Department of Agriculture Animal Welfare Act and were approved by the GlaxoSmithKline or Mispro Laboratory Animal Care and Use Committee.

In ZDF rats, continuous administration of Compound A104 (alone and in combination with exenatide (Ex4)) for 14 days resulted in a dose-dependent reduction in serum triglycerides (Figure 12). Compound A104 also dose-dependently reduced liver fat content and liver weight after 14 days (Figure 13).

Example 21: Conventional synthesis method for installing lipophilic substituents and spacers of Formula II

Lipophilic substituents and spacers of Formula II are incorporated into many of the disclosed peptides:

Wherein the indicated carbonyl group on the structure of Formula II is covalently linked to reveal the epsilon-amine group of the lysine residue of the peptide to form a amide bond.

Linear peptide sequences were synthesized by solid-phase methods on a Prelude peptide synthesizer (Protein Technologies Inc., Tucson, AZ) using the Fmoc strategy with 2-(1H-benzotriazol-1-yl)-1,1 ,3,3-tetramethylurea hexafluorophosphate (HBTU) or 2-(6-chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethylamine Onium hexafluorophosphate (HCTU) (5-fold molar excess) was activated in N,N-dimethylformamide (DMF) with N'N-diisopropylethylamine (DIEA) as base , use 20% piperidine/DMF for Fmoc deprotection. The resin was Rink Amide MBHA LL (Novabiochem) or N-α-Fmoc protected, preloaded Wang LL (Novabiochem), loaded at 0.29 to 0.35 mmol/g in the 20 to 400 μmol scale. FMOC-Lys(allyloxycarbonyl)-OH is preferentially substituted into the peptide chain at the desired acyl-chain substitution position. Next, Boc-Tyr(tbu)-OH or Boc-Trp(Boc)-OH was used as the N-terminal amino acid residue. After completing the synthesis, the resin was washed with dichloromethane (DCM) and dried under vacuum for 30 minutes. Next, the allyloxycarbonyl protecting group was removed using tetrakis(triphenylphosphine)palladium solution (in CHCl ₃ /acetic acid/N-methylmorpholine, 37:2:1 ratio). The resin was subsequently washed with a 0.5% solution of sodium diethyldithiocarbamate trihydrate in DMF, then with a 0.5% solution of DIEA in DMF, and finally with DMF. Next, FMOC-Glu(Otbu)-OH was coupled to the free amine acid side chain using normal phase solid phase conditions. C-16 acyl side chains were added using palmitic acid under normal phase solid phase method. The resin was treated with (92.5% TFA, 2.5% phenol, 2.5% water, and 2.5% triisopropylsilane) for 2 to 3 hours to allow for final deprotection and free-standing cleavage of the peptide. The cleaved peptides were precipitated using cold diethyl ether. The diethyl ether was decanted and the solid was triturated again with cold diethyl ether, centrifuged into a pellet, and freeze-dried. The freeze-dried solid was redissolved in a 1:1 acetonitrile/water solution (10 to 15 mL) containing 0.1% TFA and subjected to reverse phase HPLC on a Waters XBridge ^™ BEH 130, CI8, 10um, 130Å, 30×250mm ID column. Purification was performed using a gradient ranging from 5 to 75% acetonitrile/water (containing 0.1% TFA) over 30 minutes, flow rate 30 mL/min, λ-215 nm. Set the column heater at 60°C. The purified product was freeze-dried and analyzed by ESI-LC/MS and analytical HPLC; the results showed pure product (>98%). The quality results are consistent with the calculated values.

HPLC analysis conditions: 4.6×250mm XBridge BEH130, 5um, C18 column using analytical Agilent 1100 and the following gradient: 20% to 100% for 15 minutes and maintained at 100% to 20 minutes. The column temperature is set at 40°C. The flow rate was set at 1.0 mL/min. The solvent is composed of A=H2O+0.1%TFA and B=acetonitrile+0.1%TFA. Observe crude and final LCMS and identify product quality using the following conditions: UV detection set at 215 and 280 nm.

LCMS analysis conditions: 4.6×250mm XBridge BEH130, 5um, C18 column uses analytical Agilent 1100 combined with API-4000 Sciex LC/MS/MS system and the following gradient: 20%B to 95%B for 10 minutes, maintaining at 95% to wash the column in 1.2min . Equilibrate the column with 5%B, 95%A for 12.5 minutes. The column temperature is set at 40°C. The flow rate was set at 1.5 mL/min. The solvent is composed of A=H2O+0.1%TFA and B=acetonitrile+0.1%TFA. UV detection is set at 215 and 280nm. method=Q1MS. Syringe size 100uL, UV range 190 to 400nm, slit width = 4mm, sampling frequency => 20Hz., ion source: electrospray, polarity = positive.

Example 22: Conventional synthesis method for installing lipophilic substituents and spacers of Formula III

The lipophilic substituents and spacers of Formula III are incorporated into many of the disclosed peptides:

wherein the indicated carbonyl group on the structure of formula III is covalently linked to reveal the epsilon-amine group of the lysine residue of the peptide to form a amide bond.

Linear peptide sequences were synthesized by solid-phase methods on a Prelude peptide synthesizer (Protein Technologies Inc., Tucson, AZ) using the Fmoc strategy with 2-(1H-benzotriazol-1-yl)-1,1 ,3,3-tetramethylurea hexafluorophosphate (HBTU) or 2-(6-chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethylamine Onium hexafluorophosphate (HCTU) (5-fold molar excess) was activated in N,N-dimethylformamide (DMF) with N'N-diisopropylethylamine (DIEA) as base , use 20% piperidine/DMF for Fmoc deprotection. The resin was Rink Amide MBHA LL (Novabiochem) or N-α-Fmoc protected, preloaded Wang LL (Novabiochem), loaded at 0.29 to 0.35 mmol/g in the 20 to 400 μmol scale. FMOC-Lys(allyloxycarbonyl)-OH is preferentially substituted into the peptide chain at the desired acyl-chain substitution position. Next, Boc-Tyr(tbu)-OH or Boc-Trp(Boc)-OH was used as the N-terminal amino acid residue. After completing the synthesis, the resin was washed with dichloromethane (DCM) and dried under vacuum for 30 minutes. Next, the allyloxycarbonyl protecting group was removed using tetrakis(triphenylphosphine)palladium solution (in CHCl ₃ /acetic acid/N-methylmorpholine, 37:2:1 ratio). The resin was subsequently washed with a 0.5% solution of sodium diethyldithiocarbamate trihydrate in DMF, then with a 0.5% solution of DIEA in DMF, and finally with DMF. Next, lengthening of the spacer region was performed by coupling {2-[2-(Fmoc-amino)ethoxy}acetic acid, followed by FMOC-Glu(Otbu)-OH, using normal phase solid phase conditions. C-18 acid was added to terminate the side chains using octadecanedioic acid under normal phase solid phase method. The resin was treated with (92.5% TFA, 2.5% phenol, 2.5% water, and 2.5% triisopropylsilane) for 2 to 3 hours to allow for final deprotection and free-standing cleavage of the peptide. The cleaved peptides were precipitated using cold diethyl ether. The diethyl ether was decanted and the solid was triturated again with cold diethyl ether, centrifuged into a pellet, and freeze-dried. The freeze-dried solid was redissolved in a 1:1 acetonitrile/water solution (10 to 15 mL) containing 0.1% TFA and subjected to reverse phase HPLC on a Waters XBridge ^™ BEH 130, CI8, 10um, 130Å, 30×250mm ID column. Purification was performed using a gradient ranging from 5 to 75% acetonitrile/water (containing 0.1% TFA) over 30 minutes, flow rate 30 mL/min, λ-215 nm. Set the column heater at 60°C. The purified product was freeze-dried and analyzed by ESI-LC/MS and analytical HPLC; the results showed pure product (>98%). The quality results are consistent with the calculated values.

HPLC analysis conditions: 4.6×250mm XBridge BEH130, 5um, C18 column using analytical Agilent 1100 and the following gradient: 5% to 70% for 15 minutes and maintained at 70% to 20 minutes. The column temperature is set at 40°C. The flow rate was set at 1.5 mL/min. The solvent is composed of A=H2O+0.1%TFA and B=acetonitrile+0.1%TFA. Observe crude and final LCMS and identify product quality using the following conditions: UV detection set at 215 and 280 nm.

LCMS analysis conditions: 4.6×250mm XBridge BEH130, 5um, C18 column using analytical Agilent 1100 combined with API-4000 Sciex LC/MS/MS system and the following gradient: 5% to 65% over 10 minutes, rising to above 95% The column was washed in 1 minute and equilibrated back to 5% organic to 12.5 minutes. The column temperature is set at 40°C. The flow rate was set at 1.5 mL/min. The solvent is composed of A=H2O+0.1%TFA and B=acetonitrile+0.1%TFA. UV detection is set at 215 and 280nm. method=Q1MS. Syringe size 100uL, UV range 190 to 400nm, slit width = 4mm, sampling frequency => 20Hz., ion source: electrospray, polarity = positive.

Each epsilon-amine group of the lysine residue of the indicated peptide residue (K****) in the compounds of Tables 7 and 8 is covalently linked to the indicated carbonyl group of the structure of Formula II to form an amide:

Each ε-amine group of the lysine residue of the indicated peptide residue (K*****) in the compounds of Tables 7 and 8 is covalently linked to the indicated carbonyl group of the structure of Formula III to form an amide :

Example 23: Clearance of peptides from the kidney after intravenous infusion (CL)

The peptide was prepared in sterile saline and administered via a jugular vein cannula to non-fasted male Wistar Han or Sprague-Dawley rats (n=3 per group) as a 3-hour intravenous infusion at a final dose of 0.3 or 0.1 mg/kg. The formula is administered at a rate of 1.67 mL/kg/h. Blood samples (approximately 250uL) were collected for pharmacokinetic analysis via the femoral vein cuff. tubes and collected into microblood collection tubes containing K2EDTA as anticoagulant and 25uL of protease inhibitor cocktail at 1, 2, 3, 3.17, 3.33, 3.5, 4, 4.5, 5, and 6 hours after the start of infusion. Plasma was prepared by centrifugation and stored at -80°C until analysis.

The acylated form was prepared in sterile saline, and intravenously infused for 1 hour, and administered to non-fasted male Sprague-Dawley rats (n=3 per group) through the jugular vein cannula. The final dose was 0.033 mg/ kg. The formula is administered at a rate of 1.67 mL/kg/h. Blood samples (approximately 250uL) were collected for pharmacokinetic analysis via femoral vein cannula at 0, 0.25, 0.5, 1, 1.17, 1.33, 1.5, 2, 4, 6, 8, 24, 30 after the start of infusion. , and 48 h were collected into microblood collection tubes containing K2EDTA as an anticoagulant and 25uL of protease inhibitor cocktail. Plasma was prepared by centrifugation and stored at -80°C until analysis. Representative results are provided in Table 8 below.

Example 24: Rats after continuous administration of exenatide or glucagon analogues weight loss

Diet-induced obese LE rats with an initial weight of 586±86 (mean±SD) were each implanted with two osmotic small pumps (Alzet) to deliver exenatide or glucagon analogues, namely selective glucagon. Receptor agonists (eg, as described in 10). Treatment groups included 30 different combinations of exenatide and glucagon analogues, with 10 to 20 animals in each group.

Measure your weight every 7 days. The results are shown in a 3-D graph of weight loss in Figure 14, with the vertical (Z) axis representing the percentage of initial body weight after 21 days of continuous dosing. Positive weight loss is shown as a circle at the top of a line rising from the base, thus representing a decrease in body weight relative to pre-treatment weight. To maintain clarity, error bars are not shown.

The horizontal axis in Figure 14 relates to the logarithm of the daily dose (in micrograms/kg body weight/day) of each of the exenatide and glucagon analogues. A −1 logarithmic dose of exenatide represents a treatment with zero exenatide dose (glucagon analogue treatment only), and a −1 on the logarithmic dose axis of glucagon represents a treatment in which no glucagon was administered ( exenatide treatment only). The series of points in the left and right foreground therefore depict the dose response for exenatide only and glucagon analogue only, respectively.

Only the weight loss response after 21 days of exenatide treatment can be described by a 4-parameter S-shaped curve. The maximum response was a weight loss of 12.5%, the ED ₅₀ was 9.6 μg/kg/day, and the Hill slope was 0.88. For glucagon analogs only, the 21-day weight loss parameters were a maximum of 28.5%, an _ED50 of 45 μg/kg/day, and a Hill slope of 2.7.

Example 25: Evaluation of Exenatide/Glucagon Analog Combinations

The raw data of Figure 14 were fit to a continuous function to be able to characterize certain key features of the response to the exenatide/glucagon analog combination. The general form of a continuous response surface is the response to a combination = (a * Resp _EX ) + (b * Resp _GGN ) + (c * Resp _EX * Resp _GGN )

Among them, Resp _EX and Resp _GGN represent the dose-dependent responses observed in each single case.

The combined response was further constrained using a hyperbolic relationship so that it did not exceed 100% weight loss. The best-fitting continuous response surface (R = 0.9) was obtained using GraphPad Prism v7.0 (GraphPad software, San Diego, CA) and the least squares iterative approximation method to the above user-defined equation. When C>0, there is a multiplicative (superadditive) component.

The symbols representing each treatment group are shown in Figure 15 as spheres embedded within the best-fit surface mesh. The four dashed curves connecting the exenatide-only and glucagon analogue-only planes represent the additive interactions that would be obtained when one dose was substituted for equi-effective doses of another. Lines of Equivalent Effects (Equivalent Lines) (Showing expected equivalent lines of 2%, 4%, 6% and 8% weight loss). The four thick solid lines depict the actual observations and are the response surfaces corresponding to the dose replacement. The difference between the solid line (observed) and the dashed line (expected) defines the synergistic interaction. For each expected level of effect, a certain dose ratio will have the highest effect and greatest synergism.

Example 26: Evaluation of fixed ratio combinations of exenatide/glucagon analogues

Since combination products may contain a fixed ratio of dose, so the response surface is analyzed from the point of view of increasing dosage of the fixed ratio mixture.

Fixed dose ratios are depicted in Figure 16 as parallel diagonal planes above the base plane of the logarithm of exenatide ("EX") versus the logarithm of glucagon analog ("GGN"). The dose response for each mixture is shown as a series of black curves where the response surface intersects each plane. The four gray curves on the surface (shown as thick black lines in Figure 15) are the responses observed when one dose is substituted for an equivalent dose of another dose, called a "dose-ratio scan" ”.

The thick black line, where the fixed dose ratio gray plane intersects the response surface, appears to cross the four gray lines at their dose-ratio optima. The gray plane defines the 3:1 GGN:EX dose ratio. 10:1 and 1:1 GGN:EX also performed well.

Example 27: Assessment of efficacy in vivo

The in vivo efficacy of exenatide/glucagon analog combinations at different fixed ratios was evaluated, with in vivo efficacy defined as the effect per unit of total drug mass.

For some applications, such as those used in small osmotic pumps where drug reservoir volume is limited, the mixture with the highest apparent in vivo potency is most advantageous. The apparent in vivo potency of different mixtures can be defined as the effect per unit of total mass of drug (or more strictly speaking, per volume of formulation).

The response to the mixture, shown as the curved surfaces of Figures 15 and 16, can thereby be reduced to a 2-D dose response as shown in Figure 17. Figure 17 X The axis is not the dose of a single dose, but the mass of two doses in a fixed ratio mixture.

Dose-response families for different mixtures include those in which the minimum combined dose elicits a given effect (illustrated by a 20% weight loss, as indicated by the horizontal arrow). A promising mixture was therefore identified as a GGN:EX mixture of approximately 3:1. The reaction for the mixture exhibiting the highest apparent potency is shown as a dashed line.

Other embodiments

While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not to limit the scope of the invention as defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following requests.

<110> INTARCIA THERAPEUTICS, INC.

<120> Glucagon receptor selective polypeptides and methods of using them

<140>

<141> 2017/5/15

<150> US 62/337,005

<151> 2016/5/16

<150> US 62/414,146

<151> 2016/10/28

<150> US 62/420,937

<151> 2016-11-11

<160> 200

<170> PatentIn version 3.5

<210> 1

<211> 29

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> X is Y or W

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> X series S, G or T

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> X series Q or H

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> X is Y or H

<220>

<221> MISC_FEATURE

<222> (11)..(11)

<223> X series S or T

<220>

<221> MISC_FEATURE

<222> (12)..(12)

<223> X series K or R

<220>

<221> MISC_FEATURE

<222> (13)..(13)

<223> X is Y, L or W

<220>

<221> MISC_FEATRE

<222> (15)..(15)

<223> X series D or E

<220>

<221> MISC_FEATURE

<222> (16)..(16)

<223> X is S, Aib, A, E, L, Q, K or I

<220>

<221> MISC_FEATURE

<222> (17)..(17)

<223> X series K, E, S, T or A

<220>

<221> MISC_FEATURE

<222> (18)..(18)

<223> X is A, R, S, E, L, T or Y

<220>

<221> MISC_FEATURE

<222> (23)..(23)

<223> X series T or V

<220>

<221> MISC_FEATURE

<222> (24)..(24)

<223> X series K, I, L or Aib

<220>

<221> MISC_FEATURE

<222> (25)..(25)

<223> X series H or W

<220>

<221> MISC_FEATURE

<222> (29)..(29)

<223> The E series is optionally attached to the Z tail as defined in the specification

<400> 1

<210> 2

<211> 29

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> X is Y or W

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> X series S or G

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> X series Q or H

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> X is Y or H

<220>

<221> MISC_FEATURE

<222> (11)..(11)

<223> X series S or T

<220>

<221> MISC_FEATURE

<222> (12)..(12)

<223> X series K or R

<220>

<221> MISC_FEATURE

<222> (13)..(13)

<223> X is Y, L or W

<220>

<221> MISC_FEATURE

<222> (15)..(15)

<223> X series D or E

<220>

<221> MISC_FEATURE

<222> (16)..(16)

<223> X series Aib, A or S

<220>

<221> MISC_FEATURE

<222> (17)..(17)

<223> X series A or K

<220>

<221> MISC_FEATURE

<222> (18)..(18)

<223> X is R, S, L or Y

<220>

<221> MISC_FEATURE

<222> (24)..(24)

<223> X series K, I or Aib

<220>

<221> MISC_FEATURE

<222> (25)..(25)

<223> X series H or W

<220>

<221> MISC_FEATURE

<222> (29)..(29)

<223> The E series is optionally attached to the Z tail as defined in the specification

<400> 2

<210> 3

<211> 29

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> X series Q or H

<220>

<221> MISC_FEATURE

<222> (16)..(16)

<223> X series Aib or A

<220>

<221> MISC_FEATURE

<222> (17)..(17)

<223> X series A or K

<220>

<221> MISC_FEATURE

<222> (18)..(18)

<223> X series R, S or Y

<220>

<221> MISC_FEATURE

<222> (24)..(24)

<223> X series K or Aib

<220>

<221> MISC_FEATURE

<222> (29)..(29)

<223> E is attached to the tail of Z as defined in the instructions

<400> 3

<210> 4

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 4

<210> 5

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 5

<210> 6

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 6

<210> 7

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 7

<210> 8

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 8

<210> 9

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 9

<210> 10

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 10

<210> 11

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 11

<210> 12

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 12

<210> 13

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 13

<210> 14

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 14

<210> 15

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 15

<210> 16

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Amination

<400> 16

<210> 17

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 17

<210> 18

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Amination

<400> 18

<210> 19

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 19

<210> 20

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 20

<210> 21

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Amination

<400> 21

<210> 22

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 22

<210> 23

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 23

<210> 24

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 24

<210> 25

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 25

<210> 26

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Amination

<400> 26

<210> 27

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 27

<210> 28

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 28

<210> 29

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 29

<210> 30

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 30

<210> 31

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Amination

<400> 31

<210> 32

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 32

<210> 33

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 33

<210> 34

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 34

<210> 35

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 35

<210> 36

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 36

<210> 37

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 37

<210> 38

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 38

<210> 39

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 39

<210> 40

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 40

<210> 41

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 41

<210> 42

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 42

<210> 43

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (38)..(38)

<223> Amination

<400> 43

<210> 44

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<400> 44

<210> 45

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (38)..(38)

<223> Amination

<400> 45

<210> 46

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 46

<210> 47

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 47

<210> 48

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<220>

<221> MOD_RES

<222> (38)..(38)

<223> Amination

<400> 48

<210> 49

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 49

<210> 50

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 50

<210> 51

<211> 40

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 51

<210> 52

<211> 35

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 52

<210> 53

<211> 36

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 53

<210> 54

<211> 37

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 54

<210> 55

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 55

<210> 56

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 56

<210> 57

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 57

<210> 58

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 58

<210> 59

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 59

<210> 60

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 60

<210> 61

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 61

<210> 62

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 62

<210> 63

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 63

<210> 64

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 64

<210> 65

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 65

<210> 66

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 66

<210> 67

<211> 40

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 67

<210> 68

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 68

<210> 69

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 69

<210> 70

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 70

<210> 71

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 71

<210> 72

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 72

<210> 73

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 73

<210> 74

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 74

<210> 75

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 75

<210> 76

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 76

<210> 77

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 77

<210> 78

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 78

<210> 79

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 79

<210> 80

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 80

<210> 81

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 81

<210> 82

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 82

<210> 83

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 83

<210> 84

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 84

<210> 85

<211> 37

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 85

<210> 86

<211> 37

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 86

<210> 87

<211> 37

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 87

<210> 88

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 88

<210> 89

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 89

<210> 90

<211> 37

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 90

<210> 91

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 91

<210> 92

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 92

<210> 93

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<400> 93

<210> 94

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 94

<210> 95

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 95

<210> 96

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 96

<210> 97

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 97

<210> 98

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 98

<210> 99

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 99

<210> 100

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 100

<210> 101

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 101

<210> 102

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 102

<210> 103

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 103

<210> 104

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 104

<210> 105

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<220>

<221> MOD_RES

<222> (38)..(38)

<223> Amination

<400> 105

<210> 106

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<220>

<221> MOD_RES

<222> (38)..(38)

<223> Amination

<400> 106

<210> 107

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<220>

<221> MOD_RES

<222> (38)..(38)

<223> Amination

<400> 107

<210> 108

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<220>

<221> MOD_RES

<222> (38)..(38)

<223> Amination

<400> 108

<210> 109

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 109

<210> 110

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 110

<210> 111

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 111

<210> 112

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (38)..(38)

<223> Amination

<400> 112

<210> 113

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (38)..(38)

<223> Amination

<400> 113

<210> 114

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (38)..(38)

<223> Amination

<400> 114

<210> 115

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 115

<210> 116

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 116

<210> 117

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 117

<210> 118

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 118

<210> 119

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 119

<210> 120

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (38)..(38)

<223> Amination

<400> 120

<210> 121

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 121

<210> 122

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 122

<210> 123

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 123

<210> 124

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 124

<210> 125

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<220>

<221> MOD_RES

<222> (38)..(38)

<223> Amination

<400> 125

<210> 126

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 126

<210> 127

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 127

<210> 128

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 128

<210> 129

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 129

<210> 130

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 130

<210> 131

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 131

<210> 132

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 132

<210> 133

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 133

<210> 134

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 134

<210> 135

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 135

<210> 136

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 136

<210> 137

<211> 29

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (17)..(17)

<223> K at position 17 and E at position 21 are located in the lactam bridge

<220>

<221> MOD_RES

<222> (21)..(21)

<223> E at position 21 and K at position 17 are located in the lactam bridge

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Xaa is K or K at position 17 and E at position 28 are located in the lactam bridge

<220>

<221> MOD_RES

<222> (27)..(27)

<223> Xaa series Q or D

<220>

<221> MOD_RES

<222> (28)..(28)

<223> Xaa is E or E at position 28 and K at position 24 are located in the lactam bridge

<400> 137

<210> 138

<211> 29

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (17)..(17)

<223> K at position 17 and E at position 21 are located in the lactam bridge

<220>

<221> MOD_RES

<222> (21)..(21)

<223> E at position 21 and K at position 17 are located in the lactam bridge

<220>

<221> MOD_RES

<222> (24)..(24)

<223> K at position 24 and E at position 28 are located in the lactam bridge

<220>

<221> MOD_RES

<222> (28)..(28)

<223> E at position 28 and K at position 24 are located in the lactam bridge

<400> 138

<210> 139

<211> 29

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (17)..(17)

<223> K at position 17 and E at position 21 are located in the lactam bridge

<220>

<221> MOD_RES

<222> (21)..(21)

<223> E at position 21 and K at position 17 are located in the lactam bridge

<220>

<221> MOD_RES

<222> (24)..(24)

<223> K at position 24 and E at position 28 are located in the lactam bridge

<220>

<221> MOD_RES

<222> (28)..(28)

<223> E at position 28 and K at position 24 are located in the lactam bridge

<400> 139

<210> 140

<211> 29

<212> PRT

<213> Homo sapiens

<400> 140

<210> 141

<211> 29

<212> PRT

<213> Homo sapiens

<400> 141

<210> 142

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (39)..(39)

<223> Amination

<400> 142

<210> 143

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 143

<210> 144

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 144

<210> 145

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 145

<210> 146

<211> 29

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 146

<210> 147

<211> 29

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<400> 147

<210> 148

<211> 29

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<400> 148

<210> 149

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 149

<210> 150

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 150

<210> 151

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> Xaa is Y or W

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> Xaa series H or Q

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> Xaa is Y, K or K in which the ε -amine group of the lysine side chain is optionally covalently connected to the lipophilic substituent via a spacer as defined in the specification.

<220>

<221> MISC_FEATURE

<222> (16)..(16)

<223> Xaa is A, S, Aib, K or K in which the ε -amine group of the lysine side chain is optionally covalently connected to the lipophilic substituent via a spacer as defined in the specification

<220>

<221> MISC_FEATURE

<222> (17)..(17)

<223>

<220>

<221> MISC_FEATURE

<222> (18)..(18)

<223> Xaa is Y, S or R

<220>

<221> MISC_FEATURE

<222> (21)..(21)

<223> Xaa is E, K or K in which the ε -amine group of the lysine side chain is optionally covalently connected to the lipophilic substituent via a spacer as defined in the specification.

<220>

<221> MISC_FEATURE

<222> (24)..(24)

<223> Xaa is I, Aib, K or K in which the ε -amine group of the lysine side chain is optionally covalently connected to the lipophilic substituent via a spacer as defined in the specification

<220>

<221> MISC_FEATURE

<222> (31)..(31)

<223> Xaa is S, K or K where the ε -amine group of the lysine side chain is optionally covalently connected to the lipophilic substituent via a spacer as defined in the specification.

<220>

<221> MISC_FEATURE

<222> (33)..(33)

<223> Xaa is G, K or K in which the ε -amine group of the lysine side chain is optionally covalently connected to the lipophilic substituent via a spacer as defined in the specification.

<220>

<221> MISC_FEATURE

<222> (34)..(34)

<223> Xaa series A or S

<400> 151

<210> 152

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<220>

<221> MOD_RES

<222> (33)..(33)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula II to form an amide

<400> 152

<210> 153

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula II to form an amide

<400> 153

<210> 154

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula II to form an amide

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 154

<210> 155

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (10)..(10)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula II to form an amide

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 155

<210> 156

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<220>

<221> MOD_RES

<222> (33)..(33)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula III to form an amide

<400> 156

<210> 157

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula III to form an amide

<400> 157

<210> 158

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula III to form an amide

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 158

<210> 159

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (10)..(10)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula III to form an amide

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 159

<210> 160

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<220>

<221> MOD_RES

<222> (33)..(33)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula II to form an amide

<400> 160

<210> 161

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula II to form an amide

<400> 161

<210> 162

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula II to form an amide

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 162

<210> 163

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (10)..(10)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula II to form an amide

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 163

<210> 164

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<220>

<221> MOD_RES

<222> (33)..(33)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula III to form an amide

<400> 164

<210> 165

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula III to form an amide

<400> 165

<210> 166

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula III to form an amide

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 166

<210> 167

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (10)..(10)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula III to form an amide

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 167

<210> 168

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<220>

<221> MOD_RES

<222> (33)..(33)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula II to form an amide

<400> 168

<210> 169

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula II to form an amide

<400> 169

<210> 170

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula II to form an amide

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 170

<210> 171

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (10)..(10)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula II to form an amide

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 171

<210> 172

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<220>

<221> MOD_RES

<222> (33)..(33)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula III to form an amide

<400> 172

<210> 173

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula III to form an amide

<400> 173

<210> 174

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula III to form an amide

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 174

<210> 175

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (10)..(10)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula III to form an amide

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (24)..(24)

<223> Aib

<400> 175

<210> 176

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (33)..(33)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula II to form an amide

<400> 176

<210> 177

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula II to form an amide

<400> 177

<210> 178

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (10)..(10)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula II to form an amide

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<400> 178

<210> 179

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (21)..(21)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula II to form an amide

<400> 179

<210> 180

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (33)..(33)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula III to form an amide

<400> 180

<210> 181

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<220>

<221> MOD_RES

<222> (21)..(21)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula III to form an amide

<400> 181

<210> 182

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula III to form an amide

<400> 182

<210> 183

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (10)..(10)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula III to form an amide

<220>

<221> MOD_RES

<222> (16)..(16)

<223> Aib

<400> 183

<210> 184

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (33)..(33)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula II to form an amide

<220>

<221> MOD_RES

<222> (38)..(38)

<223> Amination

<400> 184

<210> 185

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (24)..(24)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula II to form an amide

<220>

<221> MOD_RES

<222> (38)..(38)

<223> Amination

<400> 185

<210> 186

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula II to form an amide

<220>

<221> MOD_RES

<222> (38)..(38)

<223> Amination

<400> 186

<210> 187

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (10)..(10)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula II to form an amide

<220>

<221> MOD_RES

<222> (38)..(38)

<223> Amination

<400> 187

<210> 188

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (33)..(33)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula III to form an amide

<220>

<221> MOD_RES

<222> (38)..(38)

<223> Amination

<400> 188

<210> 189

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (24)..(24)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula III to form an amide

<220>

<221> MOD_RES

<222> (38)..(38)

<223> Amination

<400> 189

<210> 190

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula III to form an amide

<220>

<221> MOD_RES

<222> (38)..(38)

<223> Amination

<400> 190

<210> 191

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (10)..(10)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula III to form an amide

<220>

<221> MOD_RES

<222> (38)..(38)

<223> Amination

<400> 191

<210> 192

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (33)..(33)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula II to form an amide

<220>

<221> MOD_RES

<222> (38)..(38)

<223> Amination

<400> 192

<210> 193

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (24)..(24)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula II to form an amide

<220>

<221> MOD_RES

<222> (38)..(38)

<223> Amination

<400> 193

<210> 194

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula II to form an amide

<220>

<221> MOD_RES

<222> (38)..(38)

<223> Amination

<400> 194

<210> 195

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (10)..(10)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula II to form an amide

<220>

<221> MOD_RES

<222> (38)..(38)

<223> Amination

<400> 195

<210> 196

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (33)..(33)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula III to form an amide

<220>

<221> MOD_RES

<222> (38)..(38)

<223> Amination

<400> 196

<210> 197

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (24)..(24)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula III to form an amide

<220>

<221> MOD_RES

<222> (38)..(38)

<223> Amination

<400> 197

<210> 198

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (16)..(16)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula III to form an amide

<220>

<221> MOD_RES

<222> (38)..(38)

<223> Amination

<400> 198

<210> 199

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<220>

<221> MOD_RES

<222> (10)..(10)

<223> K, in which the ε -amine group of the lysine acid side chain is covalently connected to the carbonyl group shown in the structure of formula III to form an amide

<220>

<221> MOD_RES

<222> (38)..(38)

<223> Amination

<400> 199

<210> 200

<211> 33

<212> PRT

<213> Artificial sequence

<220>

<223> Chemical synthesis

<400> 200

Claims (17)

_A method for preparing an isolated polypeptide _, which includes: (i) amino acid _sequence : _{X 1} X ₂ X _{3 GTFTSDX 10} X ₁₁ X _{12 X 13 LX 15} LEDE-Z tail (SEQ ID NO: 1), where: X ₁ is Y or W; X ₂ is S, G or T; X ₃ is Q or H; X ₁₀ is Y or H; X ₁₁ is S or T ; X _{12 is} K or R; X ₁₃ is Y, L or W; X ₁₅ is D or E; I; X ₁₇ is K, E, S, T or A; X ₁₈ is A, R, S, E, L, T or Y; X ₂₃ is T or V; X ₂₄ is K, I, L or Aib; X ₂₅ is H or W; and the Z tail does not exist or is selected from the group consisting of: EEPSSGAPPPS-OH (SEQ ID NO: 4); EPSSGAPPPS-OH (SEQ ID NO: 5); GAPPPS-OH (SEQ ID NO: 6); GGPSSGAPPPS-OH (SEQ ID NO: 7); GPSSGAPPPS-OH (SEQ ID NO: 8); KRNKNPPPS-OH (SEQ ID NO: 9); KRNKNPPS-OH (SEQ ID NO: 10); KRNKPPIA -OH (SEQ ID NO: 11); KRNKPPPA-OH (SEQ ID NO: 150); KRNKPPPS-OH (SEQ ID NO: 12); KSSGKPPPS-OH (SEQ ID NO: 13); PESGAPPPS-OH (SEQ ID NO : 14); PKSGAPPPS-OH (SEQ ID NO: 15); PKSKAPPPS-NH ₂ (SEQ ID NO: 16); PKSKAPPPS-OH (SEQ ID NO: 17); PKSKEPPPS-NH ₂ (SEQ ID NO: 18); PKSKEPPPS-OH (SEQ ID NO: 19); PKSKQPPPS-OH (SEQ ID NO: 20); PKSKEPPPS-NH ₂ (SEQ ID NO: 21); PSKSKSPPS-OH (SEQ ID NO: 22); PRNKNNPPS-OH (SEQ ID NO: 23); PSKGAPPPS-OH (SEQ ID NO: 24); PSSGAPPPSE-OH (SEQ ID NO: 25); PSSGAPPPS-NH ₂ (SEQ ID NO: 26); PSSGAPPPS-OH (SEQ ID NO: 27) ; PSSGAPPPSS-OH (SEQ ID NO: 28); PSSGEPPPS-OH (SEQ ID NO: 29); PSSGKKPPS-OH (SEQ ID NO: 30); PSSGKPPPS-NH ₂ (SEQ ID NO: 31); PSSGKPPPS-OH ( SEQ ID NO: 32); PSSGSPPPS-OH (SEQ ID NO: 33); PSSKAPPPS-OH (SEQ ID NO: 34); PSSKEPPPS-OH (SEQ ID NO: 35); PSSKGAPPPS-OH (SEQ ID NO: 36) ; PSSKQPPPS-OH (SEQ ID NO: 37); PSSKSPPPPS-OH (SEQ ID NO: 38); SGAPPPS-OH (SEQ ID NO: 39); and SSGAPPPS-OH (SEQ ID NO: 40); or (ii) Amino acid sequence: YSQGTFTSDYSKYLDSX ₁₇ RAQX ₂₁ FVX ₂₄ WLX ₂₇ X ₂₈ T-OH (SEQ ID _NO : 137), where _: in; X ₂₁ is E* _, where E* is _in the lactam bridge with K* at X ₁₇ ; in the amide bridge _; X ₂₇ is Q or D; _and ： _{X 1} SX _{3 GTFTSDX 10 SKYLDX 16 X 17} X ₁₈ AQX ₂₁ FVX ₂₄ WLEDEPX ₃₁ SX 33 X ₁₀ =Y, K or K***; X ₁₆ =A, S, Aib, K or K***; X ₁₇ =A, K, Aib or K***; X ₁₈ =Y, S or R ; X ₂₁ =E, K or K***; X ₂₄ =I, Aib, K _{or K***; X 31 =} S, K or K***; ; _and Steps: (a) connecting the amino acid corresponding to the C-terminal amino acid of the isolated polypeptide to the resin to generate the amino acid bonded to the resin; (b) ordering the amino acid bonded to the resin in the presence of a peptide coupling agent The amino acid reacts with an amino acid corresponding to the subsequent most N-terminal amino acid of the isolated polypeptide to generate a peptide bonded to the resin; (c) in the presence of a peptide coupling agent, the peptide bonded to the resin is ordered to react with the corresponding N-terminal amino acid. The amino acid reaction of the subsequent N-terminal amino acid of the isolated polypeptide should produce an extended peptide bound to the resin; (d) Repeat step (c) until the extended peptide bound to the resin contains the isolated polypeptide. each amino acid; and (e) cleaving the resin-bound extended peptide from the resin to provide the isolated polypeptide. The method of claim 1, wherein each amino acid in steps (a), (b) and (c) includes an Fmoc-protected amine group, and wherein in steps (a), (b) and (c) At the end of each, the Fmoc group is deprotected by piperidine. The method of claim 1, wherein each of the peptide couplers of steps (b) and (c) is selected from the group consisting of: HBTU, HCTU and HOBT. The method of claim 1, wherein steps (a), (b) and (c) further comprise adding N,N-diisopropylethylamine (DIEA). The method of claim 1, wherein the isolated polypeptide includes the amino acid sequence _: X ₁ X ₂ X ₃ GTFTSDX ₁₀ X ₁₁ X ₁₂ X ₁₃ LX ₁₅ X _{16 X 17} : 2), where: X ₁ is Y or W; X ₂ is S or G; X ₃ is Q or H; X ₁₀ is Y or H; X ₁₁ is S or _T ; X ₁₂ is K or R; It is Y, L or W; X ₁₅ is D or E; X ₁₆ is 2-aminoisobutyric acid (Aib), A or S; X ₁₇ is A or K; X ₁₈ is R, S, L or Y; X ₂₄ is K, I or Aib; and the Z tail is absent or selected from the group consisting of: PSSGAPPPS-NH ₂ (SEQ ID NO: 26); PSSGAPPPS-OH (SEQ ID NO: 27); and PKSKSPPPS- NH ₂ (SEQ ID NO: 21). The method of claim 1, wherein the isolated polypeptide includes the amino acid sequence: YSX ₃ GTFTSDYSKYLDX ₁₆ X ₁₇ X ₁₈ AQEFVX ₂₄ WLEDE-Z tail (SEQ ID NO: ₃ ), wherein: ₁₆ is 2-aminoisobutyric acid (Aib) or A; _{X 17} is A or K; X ₁₈ is R, S or Y; : 27) and PSKKSPPPS-NH ₂ (SEQ ID NO: 21). The method of claim 6, wherein the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 41 (compound A1). The method of claim 6, wherein the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 42 (compound A2). The method of claim 1, wherein the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 137. The method of claim 9, wherein the lysine residues and glutamic acid residues forming the lactam bridge respectively comprise allyloxycarbonyl (alloc) groups in their side chains when coupled to the extended peptide bonded to the resin. ) and allyl (allyl) protecting groups. The method of claim 10, wherein the allyloxycarbonyl and allyl protecting groups are removed before step (e). The method of claim 9, wherein the lactam bridge is formed before step (e). The method of claim 9, wherein the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 138 (compound A104) or the amino acid sequence of SEQ ID NO: 139 (compound A105). The method of claim 1, wherein the isolated polypeptide package Contains the amino acid sequence of SEQ ID NO: 151. The method of claim 14, wherein the lysine covalently linked to the lipophilic substituent or optionally covalently linked via a spacer, when coupled to the extended peptide bonded to the resin, includes a lysine in its side chain. Allyloxycarbonyl protected amine group. The method of claim 15, wherein the covalent linkage to the lipophilic substituent or optionally via a spacer occurs between steps (d) and (e) and includes the following steps: d.1) Remove the allyloxycarbonyl protecting group to provide a free amine side chain; (d.2) Couple the free amine side chain with Fmoc-Glu(Otbu)-OH; or make the free amine side chain The amino acid side chain is coupled to the spacer extension group {2-[2-(Fmoc-amino)ethoxy}acetic acid and the spacer extension group is subsequently coupled to Fmoc-Glu(Otbu)-OH; (d.3 ) Couple the glutamic acid residue in step (d.2) with hexadecanoic acid or octadecanoic acid. The method of claim 14, wherein each lipophilic substituent and spacer is of formula II:

Or as formula III: